The prevention of osteoporotic fracture is an essential socioeconomical priority, especially in the developed countries including Japan. Estrogen, selective estrogen-receptor modulators (SERMs), and bisphosphonate are potent inhibitors of bone resorption; and they have clinical relevance to reduce osteoporotic fractures in postmenopausal women. However, we can prevent at most 50% of vertebral fractures with these agents. For the better compliance of aminobisphosphonate, the use of a daily bisphosphonate regimen is moving to a weekly or monthly bisphosphonate regimen. Both cathepsin K inhibitors and modulators of the RANK-RANKL system, which can reduce bone resorption, are the candidates for the future treatment of osteoporosis. As well as bone resorption, we need to increase bone formation to prevent osteoporotic fractures, particularly in elderly patients with low bone turnover. In the U.S., Europe, and Australia, they have already started intermittent parathyroid hormone injection and/or oral strontium ranelate to stimulate bone formation. We still need to discover new agents to reduce osteoporotic fractures for the better quality of life without fractures.
The view that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is prevailing on the basis of successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for the signal input, Glu transporters for the signal termination and vesicular Glu transporters for the signal output through exocytotic release. Little attention has been paid to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, by contrast, whereas recent molecular biological and pharmacological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine signal mediator in bone comprised of osteoblasts, osteoclasts and osteocytes, in addition to other peripheral tissues including pancreas, adrenal and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis as an excitatory neurotransmitter in the central nervous system and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we would outline the possible signaling system for Glu to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in bone.
The vertebrate skeleton is richly innervated with adrenergic and peptidergic nerve terminals, and these play important roles in bone remodeling. Recent studies have generally shown that increased sympathetic nervous activity causes bone loss via an increase in bone resorption and a decrease in bone formation. Increased bone resorption is based on the stimulation of both osteoclast formation and osteoclast activity. These effects are associated with β2-adrenergic activity toward both osteoblastic and osteoclastic cells. Such findings indicate that β-blockers may be effective against osteoporosis, in which case there is increased sympathetic activity. This review summarizes evidence obtained both in vitro and in vivo implicating sympathetic neuron action in bone resorption.
The mass and function of bones depends on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Osteoporosis typically reflects an imbalance in skeletal turnover, such that bone resorption exceeds bone formation. Osteoclasts are target cells for anti-osteoporosis therapies. To discover new types of antiresorptive agents, we screened for natural compounds that regulate osteoclast differentiation, function, and survival. As a result, we identified reveromycin A, destruxins, mevastatin, FK506, cyclosporin A, prodigiosins, concanamycins, and symbioimine among microbial natural compounds. In this review, we discuss the mechanisms of action of these compounds on osteoclasts.
It has been reported that the pharmacological characteristics of bisphosphonates vary depending on the side chain attached to the carbon atom of the P-C-P bond. TRK-530 is a novel synthetic bisphosphonate with an anti-oxidant methylthio-phenylthio side chain. This compound has been suggested to have both anti-inflammatory and anti–bone-resorbing effects. Such a compound could be effective for the treatment of diseases with excessive bone resorption accompanied by inflammation. We have been studying this compound as a potential therapeutic agent for periodontitis. To date, we have found that 1) TRK-530 inhibited osteoclastic bone resorption in animals and in bone organ culture, 2) both systemic and topical administration of TRK-530 prevented alveolar bone loss in animals with experimental periodontitis, 3) TRK-530 prevented prostaglandin E2 synthesis by inhibiting the expression of cyclooxygenase (COX)-2 mRNA, and 4) TRK-530 inhibited the formation of dental calculus. The above results suggest that TRK-530 might be useful for the treatment of alveolar bone loss in periodontitis.
Clinical and experimental evidence suggests that glucocorticoids may be effective in the treatment of neuropathic pain, but their mechanism of action is unknown. We gave triamcinolone (3 mg/kg) to rats with an experimental post-traumatic painful peripheral neuropathy, chronic constriction injury (CCI), five days after nerve injury, when the abnormal pain syndrome is known to be present; and pain sensitivity was measured on postoperative days 7 – 14, a period during which symptoms are known to be at approximately peak severity. Additional CCI rats were treated similarly; and then they were sacrificed five days after the injection for an immunocytochemical analysis of endoneurial tumor necrosis factor-alpha (TNFα), macrophages, and mast cells in the sciatic nerve proximal to the site of injury. Vehicle-injected CCI rats demonstrated the expected neuropathic pain symptoms. Triamcinolone-treated CCI rats had a statistically significant reduction in the magnitude of heat-hyperalgesia and mechano-allodynia, but there was no effect on cold-allodynia or mechano-hyperalgesia. On the nerve-injured side of vehicle-injected rats, TNFα was present in Schwann cells and mast cells. On the nerve-injured side of triamcinolone-treated rats, there was a significant (71.5%) reduction in the number of TNFα-positive mast cells. Our results suggest that glucocorticoid therapy for neuropathic pain may work via the reduced expression of TNFα in endoneurial mast cells.
Although theophylline has been suggested to have an anti-inflammatory effect, there have been few reports to show the in vivo effect and the mechanism of anti-inflammatory activity of theophylline experimentally. To reveal the anti-inflammatory activity of theophylline, we studied the effect of theophylline and its metabolites on carrageenan-induced edema in rat foot pad. Subcutaneous injection of theophylline (5 – 100 mg/kg) inhibited carrageenan-induced edema dose-dependently. Theophylline metabolites, that is, 1-methylxanthine, 3-methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid (equimolar dose to 50 mg/kg of theophylline), did not inhibit the edema significantly. The inhibitory effect of theophylline on carrageenan-induced edema disappeared by pretreatment with aminoglutethimide, an inhibitor of glucocorticoid synthesis and with mefepristone, an antagonist of the glucocorticoid receptor. These results suggest that theophylline itself has anti-inflammatory activity and the glucocorticoid–glucocorticoid receptor system is involved in the anti-inflammatory activity of theophylline.
Reactive oxygen species have been known as important contributors to ischemia/reperfusion (I/R) injury. Studies on the beneficial effect of N-acetylcysteine (NAC), a potent antioxidant, on limiting infarct size induced by I/R yielded contrasting results. The present study was undertaken to compare the effect of NAC by different administration methods on infarct size in a rat myocardial I/R model. Rats underwent 30 min of left coronary occlusion followed by 4 h of reperfusion. Treatment with continuous infusion of NAC (150 mg/kg per hour) from 30 min before occlusion for 2 h (until 1 h after the start of reperfusion) produced a significant limitation of the infarct size as a percentage of the ischemic area (8%) compared to the non-treated control (60%). However, bolus injection of 150 mg/kg at 30 min prior to occlusion and 5 min prior to reperfusion failed to reduce it (56%) although the total dose is the same. The decreased total glutathione content and glutathione peroxidase activity in the ischemic region were recovered in the continuous infusion group, but not in the bolus injection group. The increased myeloperoxidase activity and phosphorylation of inhibitor κB after I/R were inhibited by the continuous treatment. These results indicate that the protective effect of NAC on myocardial infarction induced by I/R was different depending on the administration method. It is necessary to maintain blood concentration during the early period of reperfusion to obtain the beneficial effect of NAC.
In our previous study, anti-apoptotic effects of GABAC-receptor stimulation was suppressed by inhibitors of cAMP-dependent protein kinase (PKA), implying GABAC receptor–mediated PKA activation. The present study showed that GABAC-receptor stimulation with its agonist, cis-4-aminocrotonic acid (CACA), protected cultured hippocampal neurons from amyloid β 25 – 35 (Aβ25 – 35) peptide–enhanced glutamate neurotoxicity. This protective effect of CACA was blocked by PKA inhibitors, KT 5720 and H-89, as well as a specific GABAC-receptor antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA). To test the possibility of GABAC receptor–mediated PKA activation, association of GABAC receptor with A-kinase anchoring proteins (AKAPs) and effect of an AKAP antisense oligonucleotide on the PKA activation were examined in primary cultured rat hippocampal neurons. Stimulation of the cells with CACA-activated PKA was assessed by the phosphorylated PKA substrate (135 kDa) level. Specific antibodies raised against GABAC-receptor ρ subunits precipitated each ρ subunit, AKAP220, and PKA regulatory and catalytic subunits from rat brain lysates, suggesting that ρ is associated with the AKAP220/PKA complex. Furthermore, antisense oligonucleotide of AKAP220 suppressed such GABAC stimulation–induced PKA activation, suggesting that GABAC-receptor stimulation activates PKA via AKAP220.
We examined gastric mucosal vulnerability in a rat model of chronic obstructive pulmonary disease (COPD). Male Wistar rats were exposed to cigarette smoke for 12 weeks (CSE rats), and on the last 4 days of exposure, prednisolone was given to induce gastric mucosal injury. Histopathology, pulmonary function, arterial blood gases, and levels of lipid peroxides (LPO), prostaglandin E2 (PGE2), hypoxia-inducible factor 1 alpha subunit (HIF-1α), and vascular endothelial growth factor (VEGF) in gastric mucosa were examined. We also tested the effect of rebamipide on prednisolone-induced gastric lesions. In CSE rats, although no gastric lesions were detected, LPO, PGE2, HIF-1α, and VEGF levels were higher than in control rats. Prednisolone induced gastric hemorrhagic lesions more readily in CSE rats than controls, with concomitant decrease in PaO2 and increased levels of LPO, HIF-1α, and VEGF. Rebamipide reversed gastric lesions without affecting any parameters examined. CSE rats were found to be a useful animal model of COPD, and COPD appeared to render the gastric mucosa vulnerable to prednisolone.
Incomplete recovery of myocardial contraction after reperfusion following brief ischemia is called the “stunning phenomenon” in an animal experiment. A hydrophilic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) does not affect this phenomenon, but lipophilic statins further reduce the contraction during reperfusion. The effects of novel hydrophilic rosuvastatin and lipophilic pitavastatin on myocardial stunning in dogs were examined. In a preliminary experiment in vitro, pitavastatin reduced L6 cell viability at 10−6 M and higher, whereas rosuvastatin and pravastatin up to 10−5 M did not show such effects. An empty capsule or a capsule filled with rosuvastatin (2 mg/kg per day) or pitavastatin (0.4 mg/kg per day) was orally administered to dogs. After 3 weeks, both statins lowered the serum cholesterol level to the same extent. Under pentobarbital anesthesia, dogs were subjected to 15-min ischemia followed by 120-min reperfusion. Ischemia arrested the myocardial contraction in the ischemic area, and reperfusion recovered it but incompletely, showing the stunning phenomenon. Rosuvastatin did not modify the stunning phenomenon, while pitavastatin further deteriorated the myocardial contraction during reperfusion.
We investigated the effects of chronic simvastatin treatment on the impaired endothelium-dependent relaxation seen in aortas from type 2 diabetic mice. Starting at 8 weeks of diabetes, simvastatin (10 mg/kg per day) was administered to diabetic mice for 4 weeks. The significantly elevated systolic blood pressure in diabetic mice was normalized by simvastatin. Aortas from diabetic mice, but not those from simvastatin-treated diabetic mice, showed impaired endothelium-dependent relaxation in response to both clonidine and adrenomedullin. After preincubation with an Akt inhibitor, these relaxations were not significantly different among the three Akt inhibitor–treated groups (controls, diabetics, and simvastatin-treated diabetics). Although clonidine-induced NOx− (NO2− + NO3−) production was greatly attenuated in our diabetic model, it was normalized by simvastatin treatment. The expression levels of both total Akt protein and clonidine-induced Ser-473-phosphorylated Akt were significantly decreased in diabetic aortas, while chronic simvastatin administration improved these decreased levels. The expression level of clonidine-induced phosphorylated PTEN (phosphatase and tensin homolog deleted on chromosome ten) was significantly increased in diabetic aortas, but chronic simvastatin did not affect it. These results strongly suggest that simvastatin improves the endothelial dysfunction seen in type 2 diabetic mice via increases in Akt and Akt phosphorylation.
Verapamil, a Ca2+ entry blocker, can induce bronchorelaxation and bronchoconstriction. The mechanism of verapamil-induced bronchoconstriction is poorly understood. The present study determines the direct effect of verapamil on smooth muscle of isolated ovine airways and analyzes the mechanisms involved. Isolated tracheal strips were suspended in organ baths containing Krebs solution for isometric tension recording. Tissue responses to verapamil as assessed by basal tone were examined in the presence or absence of epithelium. The effects of verapamil on carbachol and cooling-induced contraction were also recorded. Measurement of unidirectional fluxes was carried out using 45Ca2+ in the absence or presence of verapamil. Verapamil induced contractions of basal tracheal smooth muscle that were proportional to its concentrations. Removal of epithelium did not affect the verapamil contractile effect. Verapamil-induced contractions were abolished in Ca2+-free Krebs solution containing 2 mM EGTA. Verapamil increased the 45Ca2+ influx into the tracheal smooth muscle. It caused relaxation of the muscle tone induced by carbachol or KCl, but it potentiated the effect of cooling-induced contraction. Verapamil induced Ca2+ influx that may lead to bronchoconstriction. These results proved that verapamil may worsen bronchoconstriction; therefore verapamil should be used with caution in asthmatic individuals.
To determine the mechanism(s) of the inhibitory effect of glucocorticoids on airway hyperresponsiveness in allergic bronchial asthma, the effects of systemic treatment with glucocorticoids on bronchial smooth muscle hyperresponsiveness and RhoA upregulation were investigated in rats with allergic bronchial asthma. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen. Animals were also treated with prednisolone or beclomethasone (each 10 mg/kg, i.p.) once a day during the antigen inhalation period. Repeated antigen inhalation caused a marked bronchial smooth muscle hyperresponsiveness to acetylcholine with an upregulation of RhoA. Augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain were observed in bronchial smooth muscles of the antigen-exposed animals. Systemic treatment with either glucocorticoid used inhibited the bronchial smooth muscle hypercontraction until the level of the sensitized control rats that received saline inhalation instead of antigen challenge. Interestingly, both glucocorticoids also inhibited the upregulation of RhoA and augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain. In conclusion, glucocorticoids ameliorated the augmented bronchial smooth muscle contraction by inhibiting upregulation of RhoA. These effects of glucocorticoids may account for, in part, their beneficial effects in the treatment of asthma.
Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor superfamily member, plays a major role in lipid metabolism and insulin sensitivity. We investigated the role of PPARγ in colonic epithelial cell turnover and carcinogenesis in colon because PPARγ is strongly expressed in colonic epithelium. Administration of PPARγ agonists suppressed epithelial cell turnover in mice. Expression level of β-catenin protein, a key molecule in carcinogenesis, was increased in mouse colon treated with PPARγ ligands. A direct interaction between β-catenin and PPARγ in cultured cell lines and colonic epithelium in mice was observed. Ligand-activated PPARγ ligand directly interacts with β-catenin, retaining it in the cytosol and reducing β-catenin / T cell factor (TCF) transcriptional activity that is functionally important on aberrant crypt foci (ACF) formation. PPARγ hetero-deficiency promoted the induction of ACF, but had no effect on the incidence of colonic polyps. These results indicate that PPARγ regulates colonic epithelial cell turnover via direct interactions with β-catenin, resulting in inhibition of β-catenin–mediated transcriptional pathways that are involved in promoting cell proliferation. Our findings suggest that PPARγ plays a role as a physiological regulator of colonic epithelial cell turnover and consequently predisposition to the development of colon cancer in early stage.
In the present study, we investigated the effect of adrenocorticotropic hormone (ACTH) on the immobilization of rats in the forced swim test after the administration of selegiline, a selective and irreversible monoamine oxidase (MAO)-B inhibitor. Single and repeated administration of selegiline significantly decreased the duration of immobility in normal rats. When selegiline was administered for 15 days, we observed a significant decrease in immobility in rats treated with ACTH for 14 days. The immobility-decreasing effect of selegiline was blocked by nafadotride, a selective dopamine D3–receptor antagonist in normal and ACTH-treated rats. Selegiline may be useful in an animal model of depressive conditions resistant to tricyclic antidepressant treatment via the dopamine D3 receptor.
Aripiprazole, an atypical antipsychotic drug, is a D2 dopamine–receptor partial agonist, but also has affinity to several serotonin receptors (5-HT1A,2A,2C,7). However, little is known about the contribution of serotonin receptors in the action of aripiprazole. The present study investigated the effects of aripiprazole on 5-HT2A receptor–mediated behaviors and compared them with the effects on dopamine receptor–mediated behavior in rats. Aripiprazole (10, 30 mg/kg, p.o.) inhibited the stereotyped behavior induced by apomorphine (1 mg/kg, s.c.), a dopamine-receptor agonist, and the wet-dog shake responses induced by DOI (2,5-dimethoxy-4-iodoamphetamine, 2 mg/kg, s.c.), a 5-HT2A–receptor agonist. Moreover, aripiprazole improved the disruption of prepulse inhibition induced by both apomorphine and DOI significantly. These data suggest that not only the dopaminergic system, but also the serotonergic system are involved in the antipsychotic effect of aripiprazole.
We examined the effects of Ca2+-channel blockers on sugar cataract formation in streptozotocin (65 mg/kg, i.v.)-induced diabetic rats that were given 5% D-glucose as drinking water. The diabetic rats were treated with an L-type Ca2+-channel blocker, nifedipine or verapamil, for 9 weeks from the 3rd day of streptozotocin injection. Using the full lens images of the horizontal plane captured with the new digital camera system that we developed recently, the cataract formation was quantitatively assessed in parallel with the conventional scaling method. In the animal model of diabetes mellitus, the cataracts at the peripheral region of the lens were detected 2 weeks after induction of hyperglycemia and progressed depending on the length of the diabetic period. The majority of them developed mature cataracts after 9 weeks of hyperglycemia. Nifedipine slowed the progression rate of diabetic cataracts without affecting the period of time required for the onset of this disease, whereas verapamil had no significant inhibitory effect on the diabetic cataract. These findings suggest that nifedipine may be considered as a candidate drug to suppress the progression of diabetic cataracts.
Oseltamivir, a widely used anti-influenza drug, inhibits virus neuraminidase. A mammalian homologue of this enzyme is expressed in the brain, yet the effect of oseltamivir on central neurons is largely unknown. Patch-clamp recordings ex vivo revealed that oseltamivir enhanced spike synchronization between hippocampal CA3 pyramidal cells. Time-lapse multineuron calcium imaging revealed that oseltamivir and its active metabolite evoked synchronized population bursts that recruited virtually all neurons in the network. This unique, so-far-unknown, event was attenuated by muscarinic receptor antagonist. Thus, oseltamivir is a useful tool for investigating a new aspect of neural circuit operation.
The present study examined the levels of Angiotensin II type 1 receptor (AT1) and type 2 receptor (AT2) in the brain stem and cerebral cortex of the stroke-prone spontaneously hypertensive rat (SHR-sp) after long-term treatment with three types of antihypertensive drugs: valsartan, enalapril, and amlodipine. In both tissues, expression of the AT1 was decreased by administration of each drug. Expression of the AT2 was decreased in the cerebral cortex by drug administration, but did not change in the brain stem. This study may contribute to elucidating the relationship between AT1 and AT2 expressions and the effect of antihypertensive drugs in SHR-sp brain.
The common adverse effect of centrally-injected μ-opioid receptor (μ-OR) agonists is pruritus. This study was conducted using mice to examine whether different subtypes of μ-OR would be responsible for pruritus and analgesia. Intracisternal injections of morphine and morphine-6β-glucronide (M6G), but not M3G, produced an antinociceptive effect. Morphine, but neither M6G nor M3G, induced facial scratching, a pruritus-related response. Facial scratching following morphine was not affected by the μ1-OR antagonist naloxonazine at doses that inhibited the antinociceptive effects. The results suggest that different subtype and/or splice variants of μ-OR are separately involved in pruritus and antinociception of opioids.