Effects of active vitamin D3 and parathyroid hormone on the serum osteocalcin in idiopathic hypoparathyroidism and pseudohypoparathyroidism.

Serum osteocalcin was measured in patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism, before or during the treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). Serum osteocalcin and plasma 1,25(OH)2D were decreased in 11 patients with idiopathic hypoparathyroidism before treatment (2.8 +/- 1.27 ng/ml, P less than 0.001 and 14.3 +/- 4.27 pg/ml, P less than 0.001, respectively). In 24 patients with idiopathic hypoparathyroidism during the treatment, serum osteocalcin and plasma 1,25(OH)2D were within the normal range (4.5 +/- 0.74 ng/ml and 25.7 +/- 5.69 pg/ml, respectively). In five patients with pseudohypoparathyroidism before treatment, plasma 1,25(OH)2D was decreased (15.6 +/- 10.6 pg/ml, P less than 0.001) but serum osteocalcin was normal (7.8 +/- 1.66 ng/ml). In nine patients with pseudohypoparathyroidism during the treatment with active vitamin D3, serum osteocalcin and plasma 1,25(OH)2D were normal (6.8 +/- 1.47 ng/ml and 27.2 +/- 6.0 pg/ml, respectively). Serum PTH in pseudohypoparathyroidism was increased before treatment (0.70 +/- 0.34 ng/ml, P less than 0.05) and was normal during the treatment (0.50 +/- 0.13 ng/ml). In idiopathic hypoparathyroidism, the active vitamin D3 increased serum osteocalcin without PTH. In pseudohypoparathyroidism, PTH may increase serum osteocalcin or modulate the effect of active vitamin D3 on serum osteocalcin.

Forskolin augments the effects of calcitonin on cytoplasmic spreading of isolated rat osteoclasts and plasma calcium levels in the rat.

Recently we have shown that forskolin acts synergistically with calcitonin on stimulation of cyclic AMP (cAMP) production in isolated rat osteoclasts and now show that forskolin also augments later physiological responses to calcitonin. The sensitivity of calcitonin inhibition of cytoplasmic spreading in isolated rat osteoclasts was increased 3.5-fold by simultaneous treatment with 10(-7) M forskolin, which alone had no effect on the response. In weanling rats the threshold of the hypocalcemic response to salmon calcitonin was reduced fivefold by simultaneous treatment with 200 micrograms/kg forskolin i.v., which alone did not influence the plasma calcium. These results provide further evidence that calcitonin-induced inhibition of osteoclast function is mediated by elevation of cAMP levels and suggest that forskolin could be used to augment the therapeutic effects of calcitonin.

Biphasic effect of calcitonin on tartrate-resistant acid phosphatase activity in isolated rat osteoclasts.

Tartrate-resistant acid phosphatase (TRAP) has been implicated as being involved in osteoclastic bone resorption, and calcitonin (CT) is known to inhibit the resorptive process. This study investigates the kinetics of CT action on TRAP activity in isolated rat osteoclasts using both biochemical and quantitative cytochemical methods. The latter technique has been developed to detect very small changes in intracellular TRAP activity at the single-cell level. The biochemical study showed that 10(-9) M salmon CT (sCT) decreased TRAP activity in medium throughout the experimental period; TRAP activity in the cells was increased during the first 2 h but subsequently declined and was decreased to a significant level at 6 h. TRAP activity in sCT-treated osteoclasts measured by the cytochemical method showed significant increases within the first hour. This response was dose dependent between 10(-16) and 10(-11) M sCT with EC50 at 8 X 10(-14) M. After 1 h, the initial increase in intracellular TRAP activity in CT-treated osteoclasts was followed by a decline to below control levels, reaching statistical significance at 9 h. Treatment with forskolin (10(-5) M) showed a similar trend, suggesting that this response is mediated by cyclic AMP-regulated phosphorylation events. From these results, we conclude that CT has two actions on TRAP in isolated rat osteoclasts: the first to inhibit its release, the second to inhibit its synthesis and/or increase its degradation.

Identification of plasminogen activator in osteoclasts.

Plasminogen activator (PA) was located in newborn rat osteoclasts using a single-cell assay. Immunohistochemistry using biotin-streptavidin-peroxidase indicated the presence of both tissue-type plasminogen activator (tPA) and urokinase (uPA) within the cytoplasm of osteoclasts isolated from newborn rat long bones. Electron microscopic immunohistochemistry using the biotin-streptavidin-colloidal gold system on L.R. Gold thin resin sections of undecalcified, newborn rat tibial metaphyseal trabecular bone identified these proteases in the lysosomal network of osteoclasts. uPA was also localized in marrow macrophage lysosomes, but tPA was not detected in these cells. The localization of these enzymes within osteoclasts may imply their involvement in bone resorption.

Immunoreactive corticotropin-releasing hormone, growth hormone-releasing hormone, somatostatin, and peptide histidine methionine are present in adrenal pheochromocytomas, but not in extra-adrenal pheochromocytoma.

CRH, GH-releasing hormone (GHRH), somatostatin (SRIH), and peptide histidine methionine (PHM) were measured by RIA in extracts of normal adrenal glands and in extracts from adrenal and extraadrenal pheochromocytomas. In normal adrenal glands, immunoreactive (IR) CRH, IR-SRIH, and IR-PHM were detectable, while IR-GHRH was undetectable. In all 11 cases of adrenal pheochromocytomas, the tumors contained 2 or more of these four IR-peptides. In particular, IR-CRH was found in 73% (n = 8) of adrenal pheochromocytomas, IR-GHRH in 91% (n = 10), IR-SRIH in 91% (n = 10), and IR-PHM in 82% (n = 9) of adrenal pheochromocytomas. There was no significant correlation among the concentration of these peptides in each tumor, i.e. the concentrations of the IR-peptides were independent of each other. In contrast to the adrenal pheochromocytomas, none of these 4 IR-peptides was detectable in 5 extraadrenal pheochromocytomas. Gel filtration of pooled extracts from adrenal pheochromocytomas showed that the major component of the IR-CRH, IR-GHRH, IR-SRIH, and IR-PHM eluted in the position of their synthetic counterparts. Our results suggest that 1) the normal adrenal gland contains IR-CRH, IR-SRIH, and IR-PHM, but not IR-GHRH; 2) all of the adrenal pheochromocytomas we examined contained a number of hypothalamic releasing or inhibiting hormones; 3) their tissue concentrations were independent of each other; and 4) all of the extraadrenal pheochromocytomas we examined contained no such IR-peptides. The presence of hypothalamic hormones in adrenal pheochromocytomas and their absence in extraadrenal pheochromocytomas may be due to the differences in the chromaffin cells of their origin. Our data may be helpful in the differential diagnosis between adrenal and extraadrenal pheochromocytomas.

Multiple forms of immunoreactive growth hormone-releasing hormone in human plasma, hypothalamus, and tumor tissues.

We examined the nature of GHRH in plasma of normal subjects and patients with acromegaly, hypothalamic tissue, pheochromocytoma, and GHRH-producing pancreatic tumor tissue using two RIAs of different specificity. One assay was a N-terminal assay that recognized GHRH-(1-44)-NH2, GHRH-(1-40)-OH, and GHRH-(1-37)-OH equally, and the other was a C-terminal assay that recognized only the COOH-terminal amidated sequence of GHRH-(1-44)-NH2. GHRH immunoreactivity was detectable in all samples in both assay systems, but the ratios of C- to N-terminal activity differed. The gel filtration profiles of plasma and tumor tissue revealed one peak in (or near) the position of synthetic GHRH-(1-44)-NH2. In contrast, two peaks were found in hypothalamic tissue; a major peak in the position of synthetic GHRH-(1-44)-NH2 and a higher mol wt peak. Ion exchange chromatography of the immunoreactive GHRH material from gel filtration of pooled plasma from normal subjects revealed three components of immunoreactive GHRH, one major peak in the position of GHRH-(1-40)-OH and two minor peaks in the positions of GHRH-(1-44)-NH2 and GHRH-(1-37)-OH. Two components of immunoreactive GHRH, a major peak in the position of GHRH-(1-44)-NH2 and a minor peak in the position of GHRH-(1-40)-OH, were found in hypothalamic tissue and pheochromocytomas. In the two ectopic GHRH-producing pancreatic tumors, three components of immunoreactive GHRH were detected: a major peak in the position of GHRH-(1-40)-OH, a smaller peak in the position of GHRH-(1-37)-OH, and a very small peak of GHRH-(1-44)-NH2. Synthetic GHRH-(1-44)-NH2 was not degraded by plasma during the extraction procedures. These results suggest that 1) the measured immunoreactive GHRH concentration differs when the same samples are measured by RIAs using antisera with different specificities; 2) such differences may be due to the presence of microheterogeneity of immunoreactive GHRH; 3) the microheterogeneity of immunoreactive GHRH in plasma is different from that in the hypothalamus; and 4) the posttranslational processing of GHRH in human hypothalamus is similar to that of pheochromocytomas but different from that of ectopic GHRH-producing pancreatic tumors.

Distribution, plasma concentration, and in vivo prolactin-releasing activity of peptide histidine methionine in humans.

The distribution of immunoreactive peptide histidine methionine (PHM) in human tissues and its plasma concentrations were examined using a specific RIA and gel filtration chromatography. The effects of synthetic PHM on anterior pituitary hormone secretion also were studied. Immunoreactive PHM was found in all tissues studied; high concentrations were found in the gastrointestinal tract, lung, and parotid gland. Subsequent but smaller amounts of PHM were found in the hypothalamus, pituitary stalk, olfactory lobe, and cerebral cortex. The distribution of immunoreactive PHM in human tissues was very similar to that of vasoactive intestinal polypeptide (VIP), and PHM and VIP were in equimolar concentrations. Immunoreactive PHM was also detectable in plasma of normal subjects, and similar plasma concentrations were found in patients with prolactinomas. Molecular sieve chromatography of extracts of nonneural tissues and plasma extracts revealed only one peak, eluting in the position of synthetic PHM. Two peaks of immunoreactive PHM were found in brain tissue; one coeluted with synthetic PHM, and the other eluted in the high mol wt region. Bolus injections of synthetic PHM significantly increased plasma PRL levels in a dose-dependent manner. However, PHM did not alter plasma GH, TSH, ACTH, LH, or FSH levels. These results indicate that PHM is distributed widely in human tissues, and posttranslational processing of the VIP-PHM precursor molecule may be different in different tissues. The finding of equimolar distributions of PHM and VIP is consistent with the notion that these two peptides are synthesized from a common precursor. The presence of immunoreactive PHM in human hypothalamic and pituitary stalk tissue and its specific in vivo PRL-releasing activity suggest that PHM may play an important role in the regulation of PRL secretion.

Role of vasopressin in cardiovascular response to central cholinergic stimulation in rats.

The cardiovascular effects of centrally administered cholinomimetics were examined in conscious Long-Evans and Brattleboro rats. Carbachol (1 microgram/kg) or physostigmine (50 micrograms/kg) induced a long-lasting increase in blood pressure and a decrease in heart rate in Long-Evans rats whereas no bradycardia was observed in Brattleboro rats, and the pressor response was significantly less than that in Long-Evans rats. The cardiovascular responses to nicotine (30 micrograms/kg) in Brattleboro rats were not different from those in Long-Evans rats. Intravenous vasopressin antagonist, d(CH2)5Tyr(Me) arginine vasopressin, significantly attenuated the pressor response and eliminated the bradycardic response to carbachol in Long-Evans rats. However, the pressor response to carbachol in Brattleboro rats was still significantly less than that in Long-Evans rats treated with vasopressin antagonist. Intravenous phentolamine partially inhibited the pressor response to carbachol in Long-Evans rats and completely eliminated it in Brattleboro rats. Combined intravenous treatment with phentolamine and vasopressin antagonist completely eliminated the pressor response to carbachol in Long-Evans rats. Centrally administered methylatropine eliminated either the hypertensive or bradycardic response to carbachol in Long-Evans rats. These results indicate that the pressor and bradycardic response to carbachol or physostigmine is mediated by the central muscarinic receptor mechanism. Hypertensive response to intracerebroventricularly administered carbachol in normal rats is mediated both by an increase in central sympathetic outflow and in circulating vasopressin. The bradycardia seems to be mediated mainly by vasopressin.

Sensorineural hearing loss associated with hypoparathyroidism.

The hearing loss of 21 patients with hypoparathyroidism was investigated by pure tone audiometry, short increment sensitivity index (SISI) test, Békésy audiometry, speech audiometry, and auditory brain stem response. Sensorineural hearing loss was found in 7 of 21 patients (13 of 42 ears) receiving no treatment for hypoparathyroidism or having chronic hypocalcemia. The high SISI score, presence of recruitment, and prolongation of the wave I (N1) latency suggested that the inner ear is responsible for hearing loss in these cases. Inner ear dysfunction was probably due to the low calcium level in inner ear fluid and/or the direct effect of vitamin D deficiency on the inner ear.

[Urinary excretion of hydroxyproline in parathyroid disorders, with special reference to its changes before and after parathyroidectomy in primary hyperparathyroidism].

It is possible to assess bone resorption from a determination of urinary excretion of hydroxyproline, which is the specific amino-acid of collagen. As dietary collagen affects 24-hour urinary excretion of hydroxyproline, it has been stated that the urine should be collected under a gelatin-free diet. A new sampling method was described in the present paper for the determination of hydroxyproline in urine, which could eliminate the affection of dietary collagen by simple fasting. The method was useful for the evaluation of bone metabolism in patients with parathyroid disorders. 10 patients with primary hyperparathyroidism (3 skeletal types and 7 non-skeletal types), 3 patients with idiopathic hypoparathyroidism and 5 normal subjects were studied. It was found that the urinary excretion of hydroxyproline increased at night and diminished during the day in patients with primary hyperparathyroidism as well as in normal subjects, but this diurnal rhythm was not clear in a patient with idiopathic hypoparathyroidism. A pilot study revealed that 10 g gelatin administered orally did not affect the urinary excretion of hydroxyproline after a 12-hour fast. Therefore, 2-hour urine samples (700 h-900 h) were collected, and blood samples were drawn at 800 h after a 13-hour fasting from 1800 h on the previous day to 700 h in the morning studied. The urinary excretion of hydroxyproline was expressed as follows: HOP (microgram/ml)/Cr(mg/dl). The 2-hour urinary excretion of hydroxyproline thus determined was highly correlated with that determined in 24-hour urine collected under a gelatin-free diet (r = 0.995, p less than 0.001) and with the total serum alkaline phosphatase activity (r = 0.987, p less than 0.001). The levels of 2-hour urinary excretion of hydroxyproline in normal subjects were 0.18-0.28 in range, and those in patients with the skeletal type of primary hyperparathyroidism were high. However, the levels were not always higher than those in the patients with the non-skeletal type, in which cases the 2-hour excretion of hydroxyproline was higher than 0.50 except in one patient. The 9 patients with primary hyperparathyroidism who had elevated levels of the 2-hour urinary excretion of hydroxyproline showed tetany after parathyroidectomy.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for an inhibitor of osteoclast attachment in dentinal matrix.

The ability of osteoclasts to colonize in vitro different preparations of dentin extracted with guanidinium hydrochloride (GuHCl) was studied in order to establish morphological evidence for an extractable inhibitor of osteoclast spreading within dentin. Osteoclasts were isolated from neonatal rats and seeded onto pieces of fully mineralized dentin, demineralized dentin and predentin extracted with GuHCl. Osteoclasts were also seeded onto unextracted tissues. The results were evaluated with scanning electron microscopy. Osteoclasts colonized and resorbed fully mineralized dentin, whereas clastic cells were not observed on unextracted demineralized dentin and predentin. In contrast to this, osteoclasts attached and spread on demineralized dentin and predentin extracted with GuHCl. It was concluded that the non-collagenous organic component of dentin contains an extractable inhibitor of osteoclastic attachment and spreading. It is tempting to speculate that the inhibitor may be responsible for the naturally occurring resistance of dentin to resorption.

Response of plasma 1,25-dihydroxyvitamin D in the human PTH(1-34) infusion test: an improved index for the diagnosis of idiopathic hypoparathyroidism and pseudohypoparathyroidism.

Synthetic human parathyroid hormone (1-34) (hPTH(1-34] infusion test has been utilized in the differential diagnosis of hypoparathyroidism by examining the incremental response of urinary phosphate and cyclic adenosine monophosphate (AMP). The response of plasma levels of 1,25-dihydroxyvitamin D (1,25(OH)2D) in parathyroid hormone (PTH) infusion test was studied as a new criterion for the differential diagnosis of idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism (PHP). Fourteen patients with IHP, 4 patients with PHP, and five control subjects were studied. All subjects received an intravenous infusion of 30 micrograms hPTH(1-34) over 5 minutes. The basal levels of plasma 1,25(OH)2D in patients with IHP and PHP were significantly lower than those in control subjects, but there was no significant difference between the levels in patients with IHP and in patients with PHP. The plasma levels of 1,25(OH)2D increased after the infusion of hPTH(1-34) and reached a peak 6 to 24 hours afterward. The 1,25(OH)2D increase at 24 hours afterward the infusion (delta 1,25(OH)2D) in control subjects and in patients with IHP were 18.1 +/- 3.91 (mean +/- SEM) and 24.1 +/- 2.80 pg/ml, respectively. There was no significant increase in patients with PHP (delta 1,25(OH)2D = 4.9 +/- 1.97 pg/ml). From these results, the measurement of delta 1,25(OH)2D in hPTH(1-34) infusion test is useful as a criterion for the differential diagnosis of hypoparathyroidism.

Treatment of pseudohypoparathyroidism with 1 alpha-hydroxyvitamin D3.

A maintenance dosis of 2 microgram/day of 1 alpha-OH-D3 could keep the level of serum calcium normal in all 5 cases with pseudohypoparathyroidism, in contrast with 4.0 +/- 1.26 microgram/day needed for the 11 cases with hypoparathyroidism (8 idiopathic and 3 postoperative). The conspicuous effect of 1 alpha-OH-D3 on pseudohypoparathyroidism is likely to be attributed to the fact that the unresponsiveness of bone tissue to parathyroid hormone is corrected by the action of active vitamin D.

25-Hydroxyvitamin D3 loading test in primary hyperparathyroidism, hypoparathyroidism and pseudohypoparathyroidism.

Plasma 1,25-dihydroxyvitamin D (1,25-(OH)2D) level, which is considered to be an indicator of parathyroid function, is possibly modified by the level of vitamin D. In the present study, we have investigated parathyroid function in terms of enhancement of the plasma levels of 1,25-(OH)2D after oral administration of 100 micrograms of 25-hydroxyvitamin D3 (25OHD3) in 9 cases of primary hyperparathyroidism (1 degree HPT), 7 cases of hypoparathyroidism (HP), 2 cases of pseudohypoparathyroidism (PHP) and 6 normal subjects. The plasma levels of 25-hydroxyvitamin D (25OHD) increased and reached a peak at 6-12 hours after the administration of 25OHD3. The plasma levels of 1,25-(OH)2D slightly increased but remained within the normal range after 25OHD3 administration in 3 of the normal subjects whose basal levels were rather low, but the increase in plasma 1,25-(OH)2D in control subjects was not statistically significant. In cases of 1 degrees HPT, the plasma 1,25-(OH)2D level rose significantly in all cases (P less than 0.05), although the pattern of the increase was not uniform. These increases were remarkable in the patients whose basal levels were low. On the other hand, an increase in the level was rarely observed in any of the cases of HP and in one of the cases of PHP. In another case, normocalcemic PHP, the plasma 1,25-(OH)2D level rose.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of parathyroid hormone on urinary excretion of N-acetyl-beta-D-glucosaminidase in idiopathic hypoparathyroidism and pseudohypoparathyroidism.

N-acetyl-beta-D-glucosaminidase (NAG) is a lysosomal enzyme predominantly located in renal proximal tubules. In idiopathic hypoparathyroidism (IHP), 100 Units of human PTH (1-34) increased urinary excretion of NAG from 0.029 +/- 0.027 to 0.173 +/- 0.035 U/lGF (p less than 0.05) in two patients before treatment and from 0.025 +/- 0.004 to 0.189 +/- 0.092U/lGF (p less than 0.02) in four patients during treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). In pseudohypoparathyroidism (PHP), PTH did not significantly increase the urinary excretion of NAG in one patient with before treatment (0.048 to 0.025 U/lGF) and four patients during treatment with active vitamin D3 (0.018 +/- 0.008 to 0.036 +/- 0.015 U/lGF). Increase in urinary excretion of NAG after injection of PTH may be a new indicator of renal effect of PTH.

[The parathyroid function in patients with hyperthyroidism].

The parameters of calcium metabolism were determined in 22 patients with untreated hyperthyroidism (5 males and 17 females) and 5 control subjects. Hypercalcemia was found in the patients with hyperthyroidism in comparison with the control subjects (serum Ca: 10.0 +/- 0.56 vs. 9.0 +/- 0.18, p less than 0.001 and Ca++: 5.1 +/- 0.28 vs. 4.6 +/- 0.15 mg/dl, p less than 0.001, mean +/- SD). Although the urinary excretion of calcium was decreased in many patients, abnormalities of phosphate metabolism were not found in this study. The parameters of bone resorption, urinary hydroxyproline, serum alkaline phosphatase and acid phosphatase, were increased in all patients with hyperthyroidism. Serum immunoreactive PTH was decreased (0.23 +/- 0.05 vs. 0.29 +/- 0.05 ngEq/ml, p less than 0.05). In vitamin D metabolites, 25-OH-D did not differ from the control (16.9 +/- 7.76 vs. 17.9 +/- 5.52 ng/ml), 1,25-(OH)2D showed a tendency to decrease (32.6 +/- 19.53 vs. 37.2 +/- 13.75 pg/ml) and 24,25-(OH)2D was obviously increased (5.57 +/- 3.582 vs. 1.73 +/- 0.619 ng/ml, p less than 0.001) in the hyperthyroid patients. Thus, the parathyroid function was suppressed in the patients with hyperthyroidism, and hypercalcemia in hyperthyroidism was suggested to be due to the direct action of thyroid hormone upon the bone.

[Scrutinization of the direct assay method for plasma cyclic AMP and clinical applications of nephrogenous cyclic AMP].

Several problems in the measurement of plasma cyclic AMP (PcAMP) and nephrogenous cyclic AMP were studied using a YAMASA RIA Kit (YAMASA Shoyu, Choshi, Japan). In this assay method, cAMP in plasma is directly succinylated without prior deproteinization, and then it is bound to antibody in an imidazole buffer. So far as the blood samples were obtained with EDTA-4Na at least 5.0 mM in the final concentration, PcAMP was not reduced until 16 hours after the blood samples were drawn. Even without EDTA, the reductions in PcAMP were not detected within 1 hour after the blood samples were drawn. This assay method for PcAMP showed parallelism in the dilution curve. Recovery was almost complete. Intra- and interassay variations were low. When plasma was incubated at 37 degrees C for 24 hours, PcAMP became negligible. Furthermore, the values of PcAMP measured with this direct assay system almost agreed with those obtained after the purification by deproteinization and Dowex column chromatography through an anion-exchange resin. The normal values of PcAMP were 13.6 +/- 3.62 pmol/ml [mean +/- SD, n = 43]. Nephrogenous cAMP expressed as a function of GFR never did show any negative values in various clinical situations. From the data of basal levels and the oral calcium tolerance test, nephrogenous cAMP appeared to be more useful than total urinary cAMP in the diagnosis of parathyroid disorders, especially hyperparathyroidism.