Vasoactive peptide-regulated gene expression during osteoblastic differentiation.

The formation of bone occurs via a series of events that are regulated by various hormones and cytokines. We previously reported that endothelin (ET) inhibits the mineralization by osteoblastic cells and natriuretic peptide (NP) promotes osteoblastic differentiation. Therefore, we attempted to identify the genes induced by ET or NP in mouse preosteoblastic MC3T3-E1 cells using the method known as differential display-polymerase chain reaction (DD-PCR) to understand the bone metabolism further. Consequently, we found that expression levels of mRNAs for fibronectin, type XII collagen, p160 Rho-associated kinase (ROCK), caldesmon, calpain, nucleolin and a novel gene with a zinc finger motif are downregulated in osteoblasts by ET stimuli. We also found that expression levels of mRNAs for eIF-4A and a novel gene are increased by C-type natriuretic peptide (CNP) stimuli.

Condylar bony change and craniofacial morphology in orthodontic patients with temporomandibular disorders (TMD) symptoms: a pilot study using helical computed tomography and magnetic resonance imaging.

OBJECTIVE: To investigate how condylar bony changes relate to craniofacial morphology using helical CT and MRI. DESIGN: Craniofacial morphology of orthodontic patients with condylar bony changes was compared with Japanese standard. SETTING AND SAMPLE POPULATION: The Department of Orthodontics, Niigata University School of Dentistry, Twenty-nine subjects were selected from orthodontic patients (six males and 23 females, a mean age of 18.8 +/- 6.3 years) who were diagnosed by helical CT as having condylar bony changes. EXPERIMENTAL VARIABLE: Subjects were divided into two groups: a unilateral condylar bony change group (unilateral group) (four males and nine females) and a bilateral condylar bony change group (bilateral group) (two males and 14 females). OUTCOME MEASURE: Condylar bony changes were evaluated on reconstructed coronal and sagittal CT scans. Disk positions were evaluated by MRI scans. Five linear and four angular measurements in lateral and posteroanterior cephalograms were compared with those of an age- and sex-matched 'standard population' from the Japanese standard. RESULTS: In the bilateral group, osteophyte formation and erosion were the common bony changes and were present in adult as well as juvenile subjects. In the unilateral group, flattening was the most common features and erosion was only present in subjects below 19 years. Disk displacement without reduction was seen in 90.6% of the bilateral group, and in 76.9% of the unilateral group. Retrognathic mandibles were shown in the bilateral group. All subjects exhibited a lateral shift of the menton toward the condylar bony changed side in the unilateral group. CONCLUSION: Condylar bony changes might be progressive and unstable in adults of the bilateral group as well as in juveniles of the both groups. It appears that condylar bony changes may be related to a lateral shift of the mandible and a retrognathic mandible in orthodontic patients with TMD symptoms.

Detectability of anterior displacement of the articular disk in the temporomandibular joint on helical computed tomography: the value of open mouth position.

OBJECTIVE: The purpose of this study was to establish the diagnostic reliability of anterior displacement of the articular disk in the temporomandibular joint on helical computed tomography. STUDY DESIGN: Ninety-four consecutive patients were examined through use of both computed tomography and magnetic resonance imaging. On axial computed tomography, anterior disk displacement was defined as the presence of an area of soft tissue density that was semilunar in shape and located in front of the mandibular condyle. RESULTS: With magnetic resonance imaging taken as the diagnostic gold standard in evaluation of articular disk position, the sensitivity, specificity, and accuracy for computed tomography were 91%, 100%, and 97%, respectively, in the closed mouth position and 96%, 99%, and 98%, respectively, in the open mouth position. CONCLUSIONS: The detectability on axial helical computed tomography of anterior displacement of the articular disk in the temporomandibular joint in the open mouth position was almost equal to that on magnetic resonance imaging. It is recommended that the open mouth position be added when helical computed tomography is used to evaluate patients with temporomandibular joint disease.

Ipriflavone down-regulates endothelin receptor levels during differentiation of rat calvarial osteoblast-like cells.

Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a synthetic flavonoid that has been shown to stimulate the activity of osteoblasts. We show here that ipriflavone also promotes the deposition of calcium and the formation of mineralized nodules by newborn rat calvarial osteoblast-like (ROB) cells as well as the activity of alkaline phosphatase. We reported previously that endothelin-1 inhibits the differentiation of ROB cells [Y. Hiruma et al. (1998) J. Cardiovasc. Pharmacol. 31, S521-S523]. Therefore, we examined the effects of ipriflavone on the expression of endothelin receptors in ROB cells by polymerase chain reaction-Southern blot analysis and in binding assays with 125I-labeled endothelin-1. Ipriflavone reduced levels of endothelin ETA receptors (to 48% of the control level) in ROB cells around day 7 in our standard cultures, while it had no apparent effect on the expression of the mRNA for the endothelin ETA receptor. By contrast, treatment with 10(-7) M endothelin-1 on days 6 through 9 alone suppressed mineralization by ROB cells. Ipriflavone also reduced the ability of endothelin-1 to inhibit mineralization by ROB cells. These results suggest that the acceleration of osteoblastic differentiation by ipriflavone might be due, at least in part, to a time-specific down-regulation of endothelin receptors.

Neurochemical evidence for inflammation-induced activation of the coeruleospinal modulation system in the rat.

By using the microdialysis technique, the concentration of noradrenaline (NA) in the dorsal horn during unilateral hindpaw inflammation was compared between rats receiving bilateral lesions of the locus coeruleus (LC) and non-operated control rats. Bilateral lesions of the LC were made using an anodal current one week before testing. Unilateral hindpaw inflammation was produced by a subcutaneous injection of carrageenan (6 mg in 0.15 ml saline). Under conditions of sodium pentobarbital anesthesia, the microdialysis probe was inserted into the dorsal horn either ipsilateral or contralateral to the site of inflammation. The NA concentration in the dialysate was measured by high-performance liquid chromatography with electrochemical detection. Prior to carrageenan injection, the NA level (baseline level) did not differ between the LC-lesioned and the non-operated groups. After carrageenan injection, in the non-operated rats, the NA level increased significantly compared to the baseline level only in the dorsal horn ipsilateral to the site of inflammation, but not in the dorsal horn contralateral to the site of inflammation. An increase of the NA level was not observed in the LC-lesioned rats and in rats receiving an injection of saline. The result suggests that unilateral hindpaw inflammation produces excitation of descending NA-containing neurons from the LC, resulting in an increase of the NA level in the dorsal horn ipsilateral to the site of inflammation.

A delta afferent fiber stimulation activates descending noradrenergic system from the locus coeruleus.

We compared the noradrenaline (NA) level in the dorsal horn following electrical stimulation of A delta afferent nerve fibers in the peripheral nervous system between rats with bilateral lesions of the locus coeruleus (LC) and non-operated control rats by using a microdialysis technique combined with high performance liquid chromatography. Prior to A delta afferent fiber stimulation, the NA content in the dialysate did not differ between the LC-lesioned and the control rats. During A delta afferent fiber stimulation, in the LC-lesioned rats, the NA level did not change significantly compared to that before A delta afferent fiber stimulation, whereas the NA level increased significantly in the control rats. There was a significant difference in the NA levels during A delta afferent fiber stimulation between the two groups of rats. The result suggests that descending noradrenergic neurons from the LC is involved in the increase of the NA level in the spinal cord dorsal horn produced by A delta afferent fiber stimulation.

Endothelins inhibit the mineralization of osteoblastic MC3T3-E1 cells through the A-type endothelin receptor.

We examined the effects of various endothelins on the mineralization of mouse clonal preosteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed mRNAs for endothelin (ET)-1 and the A-type receptor for ET (ETA). A pharmacological study also demonstrated the predominant expression of the ETA receptor. Northern blotting analysis revealed that ETs decreased the expression of mRNA for osteocalcin, which is a marker protein for the maturation of osteoblastic cells. ET-1 also decreased in the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner and it had an inhibitory effect even at 10(-11) M. The rank order of potency of ETs was ET-1 = ET-2 > ET-3. Brief treatment with 10(-7) M ET-1 on days 6-8 alone suppressed mineralization. ET-1 enhanced the rate of production of inositol 1,4, 5-trisphosphate (IP3) in MC3T3-E1 cells, but it had no effect on the rate of production of cAMP. Taken together, our data indicate that ET-1 might inhibit the mineralization of osteoblastic cells via an interaction with the ETA receptor, with generation of IP3 as the intracellular signal.

Effects of yohimbine on naloxone-induced antinociception in a rat model of inflammatory hyperalgesia.

Effects of the alpha2-adrenoceptor antagonist yohimbine on the antinociception produced by a low dose of naloxone were examined in a rat model of carrageenan-induced inflammation. In rats receiving saline prior to naloxone injection, the low dose of naloxone (5 microg/kg, i.p.) significantly prolonged paw withdrawal latency in response to noxious thermal stimuli for both the inflamed and the non-inflamed paws 4 h after carrageenan injection (6.0 mg in 0.15 ml saline). In rats receiving yohimbine, the low dose of naloxone failed to produce prolongation of paw withdrawal latencies 4 h after carrageenan, whereas naloxone produced antinociception 7 days after carrageenan. The results suggest that noradrenergic mechanisms are involved in naloxone-induced antinociception only in the early phase of carrageenan-induced inflammation.

Deceleration by angiotensin II of the differentiation and bone formation of rat calvarial osteoblastic cells.

We examined the effects of angiotensin II (Ang II) on the differentiation of rat calvarial osteoblastic cells and on the formation of bone by these cells. Northern blotting analysis revealed that Ang II inhibited the expression of mRNA for osteocalcin, which is a protein that is specifically expressed during maturation of osteoblastic cells. Ang II decreased the activity of alkaline phosphatase, a marker of osteoblastic differentiation, in the cells, acting via the type 1 (AT1) receptor. We used von Kossa staining to examine the formation of mineralized nodules by osteoblastic cells. Both the number and the total area of mineralized nodules were quantified and shown to be decreased by 10(-7) M Ang II. The accumulation of calcium in cells and the matrix layer was also decreased by Ang II. Binding analysis with subtype-specific antagonists revealed the presence of AT1 receptors for Ang II in this culture system. Ang II caused a marked increase in the rate of production of intracellular cAMP in this system. Our data suggest that Ang II might be intimately involved in osteoblastic metabolism through its interaction with the AT1 receptor.

Endothelins inhibit mineralization of rat calvarial osteoblast-like cells.

We examined the effects of members of the endothelin (ET) family on mineralization of rat calvarial osteoblast-like cells. The accumulation of calcium in cells and cell layers was attenuated by ETs with the rank order of potency ET-1 = ET-2 > ET-3. We stained the mineralized nodules by von Kossa staining and measured the number and area of mineralized nodules. The inhibitory effects of ET-1 and ET-2 on the formation of mineralized nodules were stronger than those of ET-3. Our data suggest that ET-1 may inhibit the mineralization process of osteoblastic cells through the ETA receptor.

The subnucleus reticularis dorsalis is involved in antinociception produced by a low dose of naloxone during carrageenan-induced inflammation.

The present study was designed to investigate a role of the subnucleus reticularis dorsalis (SRD) in the analgesia produced by a low dose of naloxone during carrageenan-induced inflammation. Male Sprague-Dawley rats were divided into the following two groups: (1) rats with bilateral lesions of the SRD (n = 13) and 2) sham-operated rats (n = 24). In each group, effects of a low dose of naloxone (5 microg/kg, i.p.) on thermal nociception were examined 4 h, 7 and 28 days after the induction of unilateral inflammation. Carrageenan (6 mg in 0.15 ml saline) was injected subcutaneously into the plantar surface of the left hindpaw. The analgesic effect was assessed by prolongation of the paw withdrawal latency (PWL) to heating. Prior to carrageenan injection, a low dose of naloxone did not prolong PWLs in either group. Four hours after carrageenan, a low dose of naloxone produced a prolongation of PWLs in both sham-operated and SRD-lesioned rats. Seven days after carrageenan, naloxone failed to produce analgesia in the SRD-lesioned rats but did produce analgesia in the sham-operated rats. At 28 days, a low dose of naloxone induced hyperalgesia in the inflamed paw of both groups, whereas naloxone was ineffective in the contralateral non-inflamed paw. These results suggest that the SRD plays a role in naloxone-induced analgesia during the subacute phase of inflammation (e.g. 7 days after induction of inflammation).

Angiotensin II stimulates the proliferation of osteoblast-rich populations of cells from rat calvariae.

We examined the effects of angiotensin II (Ang II) on the proliferation of osteoblast-rich populations of cells obtained from calvariae of newborn rat. Addition of Ang II to the culture medium caused dose-dependent increases in the rate of DNA synthesis. Such increases were completely inhibited by the addition of DuP753, an antagonist of AT1 receptor. Ang II potentiated the production of inositol 1,4,5-trisphosphate (IP3) in the culture. Ang II also stimulated the activities of mitogen-activated protein kinases (MAPKs) upon binding to the AT1 receptor. These results suggest that Ang II might be intimately involved in the proliferation of the cells in calvariae through the AT1 receptor.

Stimulation by retinoids of the natriuretic peptide system of osteoblastic MC3T3-E1 cells.

The effects were examined of treatment with retinoids of osteoblastic MC3T3-E1 cells on the natriuretic peptide system that promotes the differentiation of osteoblastic cells. Northern blot analysis revealed high levels of mRNA for the retinoid X receptor beta (RXR beta) and moderate levels of mRNAs for retinoic acid receptors alpha (RAR alpha) and gamma (RAR gamma). Exposure of MC3T3-E1 cells to 1 microM retinoid caused increases in the levels of C-type natriuretic peptide (CNP) and natriuretic peptide receptor-C (NPR-C). The activity of natriuretic peptide receptor-B (NPR-B) was unchanged after the addition of retinoid to the culture system. These results suggest that retinoids might influence the metabolism of osteoblastic cells through regulation of the natriuretic peptide system.

Stimulation by C-type natriuretic peptide of the differentiation of clonal osteoblastic MC3T3-E1 cells.

We examined the effects of C-type natriuretic peptide (CNP) and B-type natriuretic peptide receptor (NPR-B) system, which stimulates the intracellular production of cGMP, on osteoblastic differentiation using clonal murine calvarial MC3T3-E1 cells. CNP-like immunoreactivity was detected in the conditioned medium and in lysates of MC3T3-E1 cells. Exposure of cells to CNP caused an increase in the intracellular production of cGMP and the increase was dose-dependent, while ANP had no effect. These results imply that CNP regulates osteoblastic metabolism via NPR-B in an autocrine manner. Northern blot analysis revealed that treatment of MC3T3-E1 cells with CNP increased the steady-state levels of mRNAs for type-I collagen, cellular alkaline phosphatase (ALPase), and osteocalcin, which are well known as markers of osteoblastic differentiation. Our observations suggest the possibility that CNP functions as a local regulator of osteoblastic differentiation, acting via a cGMP-mediated pathway.

Reciprocal regulation by cyclic nucleotides of the differentiation of rat osteoblast-like cells and mineralization of nodules.

The effects of cAMP and cGMP on the differentiation of osteoblast-like cells derived from rat calvariae and on the formation of bone in vitro were studied. Continuous culture of osteoblast-like cells in the presence of 8-bromo-cyclic AMP (8-Br-cAMP) resulted in the dose-related inhibition both of the synthesis of cellular alkaline phosphatase (ALPase), which is known as a marker of osteoblastic differentiation, and of the formation of mineralized nodules, which is a model of the formation of bone in vitro. By contrast, 8-bromo-cyclic GMP (8-Br-cGMP) promoted the synthesis of ALPase and the formation of mineralized nodules. Northern blot analysis revealed that these cyclic nucleotides modulated the steady-state levels of mRNAs for ALPase and osteocalcin, a bone-matrix protein that is specifically produced by osteoblast. The present results indicate that cAMP and cGMP act reciprocally to regulate osteoblastic differentiation and the subsequent formation of mineralized nodules.