Shosei Matsumoto

Effects of retinoic acid on bone formation and resorption in cultured mouse calvaria.

1. The effects of retinoids on bone metabolism were examined in newborn mouse calvaria. 2. Incubation of calvaria with 0.01-1 microM retinoic acid for 4 days decreased their alkaline phosphatase (ALP) activity, mineral content and collagen content in a concentration-dependent fashion. 3. With treatment for 2 days, retinoic acid (1 microM) decreased the ALP activity and collagen content, but not the mineral content. 4. All these inhibitory effects were observed in calvaria from 0-day-old mice, but no inhibition of ALP activity was observed in calvaria from 14-day-old mice. 5. 1-Hydroxyethylidene-1,1-bisphosphonate (HEBP, 1 mM), which inhibits bone resorption, prevented the effect of retinoic acid (1 microM) on the bone mineral content, but not the effects on ALP and collagen (synthesized by osteoblasts). HEBP (1 mM) alone had no effect on the calvarial mineral and collagen contents. 6. These findings indicate that retinoic acid both stimulates bone resorption and inhibits osteoblastic activity by different mechanisms, and that stimulation of bone resorption by retinoic acid is inhibited by HEBP.

Alteration of in vitro bone metabolism and tooth formation by zinc

1. The effects of zinc on bone metabolism and tooth formation was examined in organ cultures of calvaria and tooth germ, and in cell cultures of osteoblast-like cells, MC3T3-E1. 2. Treatment of calvaria with zinc (10, 100 microM) for 4 days both increased alkaline phosphatase (ALP) activity in bone and reduced the secretion of N-acetyl beta-glucosaminidase from bone, without affecting bone mineral or collagen content. The increase in ALP activity produced by zinc (10 microM) was inhibited neither by actinomycin D (5 micrograms/ml) nor by cycloheximide (0.5 micrograms/ml). 3. Treatment of MC3T3-E1 cells with zinc (50, 100 microM) for 25 days also increased ALP activity, but reduced calcium content in cells and in the matrix layer. 4. These results indicate that zinc increases ALP activity in osteoblasts without affecting de novo enzyme synthesis, and that it inhibits bone mineralization, in accordance with the inhibition of osteoclastic activity. 5. Treatment of tooth germ with zinc (100 microM) for 7 days also produced an increase in ALP activity and inhibition of mineralization. These results indicate that the increased ALP activity produced by zinc is a common phenomenon in hard tissues, and, further, that zinc inhibits mineralization during tissue formation.

Alteration of Catecholamine Phenotype in Transgenic Mice Influences Expression of Adrenergic Receptor Subtypes

Abstract: Agonist‐induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N‐methyltransferase (PNMT) under the control of a human dopamine β‐hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high‐level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6–87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (−)‐[125I]iodocyanopindolol to determine changes in binding sites of β‐AR subtypes. In transgenic mice, the number of β2‐AR binding sites was 56.4–74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the β1‐AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down‐regulation of β2 binding sites was accompanied by a significant decrease in steady‐state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the β‐AR subtypes in their target tissues.

Origin of Cyclic Adenosine Monophosphate in Saliva

The level of cyclic adenosine monophosphate (AMP) in duct saliva from the dog submandibular gland was increased after cyclic AMP was administered intravenously in vivo. Isoproterenol increased the level of cyclic AMP in plasma and saliva in vivo and in salivary gland slices in vitro, but increased the level only slightly in saliva in a perfused dog submaxillary gland.

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.

Morphological differences in the skull of ascorbic acid-deficient ODS rats

The physiological importance of ascorbic acid (AsA) in bone formation has been thought to be due mainly to its effects on collagen production and, consequently, matrix formation. However, effects of AsA deficiency on growing animals are poorly understood. The purpose of this experiment was to find how AsA deficiency affects craniofacial growth cephalometrically, and the periodontal ligament and the bone density of alveolar septum histologically. Five-week-old growing male rats with hereditary defects in AsA synthesis (ODS rats) placed on an AsA-deficient or minimally supplemented diet for 4 weeks showed a marked reduction in plasma AsA level and a negative or slight increment in body weight. Cephalometric evaluation revealed that craniofacial growth was influenced by AsA deficiency. Histological observations of periodontal structure and bone density in AsA-deficient animals showed no difference from controls, whereas the periodontal ligament of the upper first molar in the deficient animals was narrower than that of controls. These observations indicate that AsA deficiency reduces craniofacial size and the width of the periodontal ligament significantly in growing rats, whereas bone density is not affected. Thus, AsA, through collagen production, may be important to the increase in size of the craniofacial structures in growing animals.

In vivo and in vitro study of the effects of chlorpromazine on tooth mineralization in rats and mice

The effects of chlorpromazine (CPZ) on tooth mineralization were examined using incisor dentine in adult rats and cultured tooth germs of mandibular first molars dissected from mouse embryos. CPZ (10, 50 and 250 mg/kg, s.c.) substantially inhibited dentine mineralization as evaluated by contact microradiographs. Plasma calcium and phosphorus concentrations were not decreased by CPZ (10 and 50 mg/kg). Physicochemical effects were not involved in the action of CPZ on the mineralization. In vitro experiments showed that CPZ (1 and 10 microM) inhibited mineralization and alkaline phosphatase (ALP) activity in the tooth germs. As CPZ has the properties of a calmodulin antagonist, the calmodulin antagonists W-7 and W-5 were also examined. Both inhibited mineralization and ALP activity in tooth germs; W-5 had less effect than W-7. These in vivo and in vitro findings suggest that CPZ inhibited cell-mediated mineralization in dentine without affecting the calcium-regulating system and physicochemical mineral deposition. In addition, calmodulin could be involved in cell-mediated mineralization.

Acute effect of parathyroid hormone-(1–34) fragment on blood pressure in rats fed a low calcium diet

1. Acute effects of human PTH-(1-34) fragment on blood pressure were examined in rats fed a low or normal calcium diet. 2. Plasma biochemistry showed that a low calcium diet evoked hyperparathyroidism as well as hypocalcemia in rats. 3. Subcutaneous administration of human PTH-(1-34) caused a significant decrease in tail-cuff systolic blood pressure in rats fed a normal calcium diet, but not in rats fed a low calcium diet. 4. Intravenous administration of human PTH-(1-34) reduced the arterial blood pressure in rats, dose-dependently. The reduction of blood pressure was more pronounced in rats fed a normal calcium diet than that in the rats fed a low calcium diet. 5. It is suggested that the hypotensive action of human PTH-(1-34) was more pronounced in the rats fed a normal diet than in the rats fed a low calcium diet, and that PTH has different cardiovascular consequences under chronic hyperparathyroidism and/or hypocalcemia.

Bone disorder in cardiomyopathic hamsters

Bones of cardiomyopathic hamsters (UM-X7.1 Syrian hamsters), at 5, 10 and 20 weeks of age, were compared chemically and histomorphologically with those of normal Syrian hamsters. Femurs of UM-X7.1 hamsters were significantly shorter than those of normal hamsters, and the mean dry weight, mean volume, mean ash weight per unit bone volume and mean ash as a percentage of dry weight of femurs were all significantly less in UM-X7.1 hamsters. The bone disorder preceded the myocardial calcium precipitation and myocardial hypertrophy in the cardiomyopathic hamsters. In addition, the percentage of cortical area measured on the cross-section of tibia and the appositional rate of bone minerals, determined by a tetracycline labelling technique, were also lower in the UM-X7.1 hamsters. These findings suggest that the bone disorder was associated with decreased bone formation in the UM-X7.1 Syrian hamsters.

Atmospheric nuclear weapon test history as characterized by the deposition of 14C in human teeth.

The 14C concentration in the collagen of human teeth was retrospectively investigated to determine whether its incorporation was related to atmospheric testing of nuclear weapons. Teeth were extracted for dental therapy from July 1987 to February 1988 from patients who were residents in Japan. Tooth collagen was extracted with HCl and converted to amorphous C by heating in a vacuum line. Specimens for 14C analysis were prepared by mixing the amorphous C with silver powder. The 14C concentration was measured by mass spectrometer. The 14C concentration in tooth collagen rapidly increased in 1961 after the bomb tests, peaked around 1967-1968, and then gradually decreased. The collagen of human teeth maintains the 14C concentration at the age of root completion for life. The results of this study indicate that the history of environmental contamination from atmospheric nuclear weapons tests has been characterized by deposition of 14C in the tooth collagen 14C of human beings.