Epileptogenesis is a big challenge. Various experimental and human studies suggested that the homeostasis of trace elements, electrolytes, membrane lipid peroxidation, and antioxidants is crucial for brain function, and they were directly or indirectly implicated as taking part in the pathophysiology of neuronal excitability, neuronal excitotoxicity, and seizure recurrence and its resistance to treatment with antiepileptic drugs (AEDs). In addition, AEDs can also alter the homeostasis of trace elements, electrolytes, and seriously increase membrane lipid peroxidation at the expense of protective antioxidants, leading to an increase in seizure recurrence and an idiosyncratic drug effect. Differential effects were detected among different AEDs treatments in which carbamazepine (CBZ) was found to be better anticonvulsant for the control of free radical related seizures and the level of trace elements were better regulated with CBZ than with valproate (VPA) and phenytoin (PHT) therapies. It is concluded that adequate trace elements and antioxidants supply is important for brain functions and prevention of neurological diseases and further elucidation of the pathological actions of such substances in the brain should result in new therapeutic approaches. Trace elements and antioxidant might have neuroprotective biological targeted benefits when used in epileptic patients.
Δ9-Tetrahydrocannabinol (Δ9-THC), the major psychoactive component of marijuana, induces catalepsy-like immobilization and impairment of spatial memory in rats. Δ9-THC also induces aggressive behavior in isolated housing stress. These abnormal behaviors could be counteracted by SR141716A, a CB1 cannabinoid receptor antagonist. Also Δ9-THC inhibited release of glutamate in the dorsal hippocampus, but this inhibition could be antagonized by SR141716A in an in vivo microdialysis study. Moreover, NMDA and AMPA-type glutamate receptor enhancers improved the Δ9-THC-induced impairment of spatial memory. On the other hand, Δ9-THC markedly inhibited the neurodegeneration in experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis and reduced the elevated glutamate level of cerebrospinal fluid induced by EAE. These therapeutic effects on EAE were reversed by SR141716A. Taken together, our results demonstrate that the inhibition of glutamate release via activation of the CB1-cannabinoid receptor is one mechanism involved in Δ9-THC-induced impairment of spatial memory, and the therapeutic effect of Δ9-THC on EAE, and a Δ9-THC analog might provide an effective treatment for psychosis and neurodegenerative diseases.
2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors. To date, two types of cannabinoid receptors (CB1 and CB2) have been identified. The CB1 receptor is assumed to be involved in the attenuation of synaptic transmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of inflammatory cells and immunocompetent cells, have not yet been fully elucidated. Recently, we investigated in detail possible physiological roles of the CB2 receptor and 2-arachidonoylglycerol in inflammation. We found that 2-arachidonoylglycerol induces the activation of p42/44 and p38 mitogen-activated protein kinases and c-Jun N-terminal kinase; actin rearrangement and morphological changes; augmented production of chemokines in HL-60 cells; and the migration of HL-60 cells differentiated into macrophage-like cells, human monocytes, natural killer cells, and eosinophils. We also found that the level of 2-arachidonoylglycerol in mouse ear is markedly elevated following treatment with 12-O-tetradecanoylphorbol 13-acetate, which induces acute inflammation. Notably, the inflammation induced by 12-O-tetradecanoylphorbol 13-acetate was blocked by treatment with SR144528, a CB2-receptor antagonist. Similar results were obtained with an allergic inflammation model in mice. These results strongly suggest that 2-arachidonoylglycerol plays essential roles in the stimulation of various inflammatory reactions in vivo.
Cannabis consumption may induce psychotic states in normal individuals, worsen psychotic symptoms of schizophrenic patients, and may facilitate precipitation of schizophrenia in vulnerable individuals. Recent studies provide additional biological and genetic evidence for the cannabinoid hypothesis of schizophrenia. Examinations using [3H]CP-55940 or [3H]SR141716A revealed that the density of CB1 receptors, a central type of cannabinoid receptor, is increased in subregions of the prefrontal cortex in schizophrenia. Anandamide, an endogenous cannabinoid, is also increased in the CSF in schizophrenia. A genetic study revealed that the CNR1 gene, which encodes CB1 receptors, is associated with schizophrenia, especially the hebephrenic type. Individuals with a 9-repeat allele of an AAT-repeat polymorphism of the gene may have a 2.3-fold higher susceptibility to schizophrenia. Recent findings consistently indicate that hyperactivity of the central cannabinoid system is involved in the pathogenesis of schizophrenia or the neural mechanisms of negative symptoms.
Growing evidence on the involvement of cannabinoids in the rewarding effects of various kinds of drugs of abuse has suggested that not only the classical dopaminergic and opioidergic, but also the most recently established endocannabinoid system is implicated in the brain reward system. Furthermore, the interplay between the three systems has been shown to be an essential neural substrate underlying many aspects of drug addiction including craving and relapse. Relapse, the resumption of drug taking following a period of drug abstinence, is considered the main hurdle in treating drug addiction. Yet, little is known about its underlying mechanisms. The link between the endocannabinoid system and the arachidonic cascade is currently being clarified. While several findings have, indeed, shown the essential role of the endocannabinoid system in the reinstatement model, the endocannabinoid-arachidonic acid pathway may also be an important part in the neural machinery underlying relapse. This evidence may provide an alternative approach that will open a novel strategy in combating drug addiction.
The distribution and characteristics of adrenomedullin (AM)-containing perivascular nerves in the rat mesenteric artery were investigated using immunohistochemical techniques. Many fibers containing AM-like immunoreactivity (LI) were observed in the adventitia of mesenteric arteries, which were densely innervated by calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-LI fibers. AM-LI, CGRP-LI, and NPY-LI fibers were abolished by cold storage denervation. Capsaicin pretreatment abolished AM-LI and NPY-LI fibers but not NPY-LI fibers. NPY-LI fibers but not AM-LI and CGRP-LI fibers disappeared after treatment with 6-hydroxydopamine. There were many AM-LI positive cells in the dorsal root ganglia, where AM mRNA was detected. In a double immunofluorescence study, AM-LI was found in CGRP-LI fibers, although some fibers contained AM-LI alone. The density of AM-LI fibers was lower in SHR than in WKY mesenteric arteries. These results suggest that the mesenteric artery is innervated by AM-containing perivascular nerves and AM may have a neurotransmitter role in the regulation of vascular tone.
Nitric oxide (NO) has many physiological functions. It is believed to be produced from L-arginine by nitric oxide synthase (NOS), and nitrite and nitrate are waste forms of it. By the way, nitrate and nitrite are abundant in vegetables and fruits, especially leafy vegetables and pickled vegetables. Orally-ingested nitrate is changed to nitrite by micro-organelles living in the hypopharynx area, and nitrite is expected to change to NO in the stomach due to its low pH. Indeed, some researchers reported that NO is produced in the gastric cavity, although few reports mentioned the physiological meanings of this NO formation. Therefore, we investigated whether the nitrite-derived NO can shift to the circulation and acts like NOS-derived NO does in tissues. We adopted a stable isotope of nitrite (15NO2−) in order to distinguish between the endogenous nitrite and the exogenously administered one and measured nitrosyl hemoglobin (HbNO) as an index of circulating NO using electron paramagnetic resonance spectroscopy. It appeared that the oral administration of 15N-nitrite formed the Hb15NO in rat blood and decreased the blood pressure of chronic L-NAME treated rats. Our findings suggest that the intake of nitrite (or nitrate)-rich foods such as vegetables and fruits would alter the systemic HbNO dynamism, resulting in the improvement of cardiovascular diseases.
It is tempting to speculate that increased vasoconstriction and loss of endothelium-dependent vasodilation might be etiological factors of elevated blood pressure in the insulin-resistant state. Vascular contraction induced by angiotensin II and the expression of NAD(P)H oxidase were increased in the aorta of insulin-resistant mice. In addition, both angiotensin II type 1 receptor expression and superoxide anion production were up-regulated in these mice. Another mechanism for imparing endothelial function is the uncoupling of endothelial nitric oxide synthase (eNOS). It has become clear from studies on the aorta of insulin-resistant rat that insulin resistance may be a pathogenic factor for endothelial dysfunction through impaired eNOS activity and increased oxidative breakdown of NO (nitric oxide) due to an enhanced formation of superoxide anion (NO/superoxide anion imbalance), which are caused by relative deficiency of tetrahydrobiopterin, a cofactor of NOS, in vascular endothelial cells. Supplementation of tetrahydrobiopterin restored endothelial function and relieved oxidative tissue damage through activation of eNOS in those rats. These results indicate that generation of superoxide anion from NAD(P)H oxidases and an uncoupled eNOS may be pathogenic factors for impaired endothelial function and hypertension in the insulin-resistant state.
The candidate mechanisms for maintaining hypertension in a chronically angiotensin II (Ang II)-infused state include direct vasoconstriction of the vasculature, disturbance of renal water/sodium handling, and central/peripheral sympathetic nerve regulation of hemodynamics. The involvement of reactive oxygen species (ROS) has been studied in these proposed mechanisms and the importance of ROS in progression of Ang II-induced hypertension has been accepted. We recently reported ROS-sensitive blood pressure regulation in chronically as well as acutely Ang II-infused hypertensive rats. The facts suggested that mechanisms for maintaining high peripheral vascular resistance in chronically Ang II-infused hypertensive rats were different from those involved in the acute hypertensive response to Ang II from the perspective of ROS sensitivity and that there must be a time-dependent transition from ROS-non-sensitive to ROS-sensitive vasoconstriction during prolonged Ang II infusion. In this review, we introduced our recent work describing the time transition of ROS sensitivity in Ang II-induced hypertension and activation of cardiovascular mitogen-activated protein kinase (MAPK) in acute and chronic phases Ang II infusion in conscious rats.
Nitric oxide (NO) from the endothelial NO synthase (eNOS) is believed to be implicated in the development and progression of atherosclerosis. The impaired endothelium-dependent vasodilatory response (EDR) has been demonstrated in vessels exposed to hypercholesterolemia and atherosclerosis. The extent of impairment serves as a predictor of future progression of atherosclerosis. As to the mechanisms of impaired EDR, increased production of superoxide is important. Recently it was revealed that eNOS becomes dysfunctional and produces superoxide rather than NO under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a co-factor for eNOS, are deficient or lacking. Dysfunctional eNOS is closely implicated in the endothelial dysfunction represented by impaired EDR in various vascular disorders including atherosclerosis. Regarding the role of eNOS in atherogenesis, experimental studies in vitro have revealed that NO from eNOS constitutes as an anti-atherogenic molecule. In eNOS-knockout mice, eNOS deficiency augments atherosclerotic lesion formation, although the effects may be partly due to the associated hypertension. However, in eNOS-transgenic mice (eNOS-Tg) crossbred with apolipoprotein E-deficient mice (apoE-KO/eNOS-Tg), we found the accelerated lesion formation in association with increased superoxide production from vessels compared with apoE-KO mice. The vascular tissue levels of BH4 were reduced and BH2, an oxidized form, levels were increased. Chronic administration of exogenous BH4 or overexpression of GTPCH-1, a rate limiting enzyme for BH4 synthesis, restored the lesion to the levels comparable to apoE-KO mice. Therefore, eNOS may have two faces in the pathophysiology of atherosclerosis depending on tissue BH4 levels.
The present study was attempted to investigate the effect of puerarin, an isoflavone compound isolated from Pueraria lobata, on both the basal body temperature and pyrogenic fever in unanesthetized, restrained rats. Intraperitoneal administration of puerarin or crude extracts of Pueraria lobata elicited hypothermia. Direct administration of a small amount of puerarin into the lateral cerebral ventricle produced the same extent of hypothermia. Systemic or central administration of puerarin causes a decrease in both colonic temperature and hypothalamic 5-HT efflux in rats. The puerarin-induced hypothermia and decreased 5-HT efflux in the hypothalamus were attenuated by selective depletion of hypothalamic 5-HT produced by intracerebroventricular injection of 5,7-dihydroxytryptamine. Furthermore, the puerarin-induced hypothermia was almost completely abolished by treatment with a 5-HT2A-receptor agonist (DOI or quipazine) or a 5-HT1A-receptor antagonist [(−)-pindolol]. A 5-HT2A-receptor antagonist (ketanserin) or a 5-HT1A-receptor agonist (8-OH-DPAT) had additive effects with puerarin. Intracerebroventricular administration of interleukin-1 caused an increase in both colonic temperature and hypothalamic 5-HT efflux. The interleukin-1-induced hyperthermia and increased 5-HT efflux in the hypothalamus were attenuated by treatment with systemic administration of puerarin. The data indicate that puerarin exerts its hypothermic and antipyretic effects by activating 5-HT1A receptor and/or antagonizing 5-HT2A receptors in the hypothalamus.
To investigate the effect of Pueraria mirifica (P. mirifica) containing phytoestrogens on reproductive systems, both sexes of rats were gonadectomized and treated orally with 0, 10, 100, and 1,000 mg/kg BW per day of P. mirifica suspended in water (abbreviated as P-0, P-10, P-100, and P-1000), respectively. The treatment schedule was separated into 3 periods: pre-treatment, treatment, and post-treatment. The duration for each period was 14 days. Blood samples were taken once a week. Serum LH and FSH levels were significantly increased within 1 week after gonadectomy; and there were no changes after administration of P-0, P-10, and P-100. However, the increase of LH levels in both sexes and FSH levels in females were attenuated within 1 week after P-1,000 treatment. The attenuation of LH levels in males was smaller than that of females. The decrease of gonadotropin levels was recovered within 1 week in males and 2 weeks in females, respectively, during the post-treatment period. The increase of uterine weight and vaginal cornification were observed in female rats treated with P-100 and P-1,000, whereas only the increase of epididymis weight was found in male rats treated with P-1,000. From this study, it can be concluded that P. mirifica can influence the reproductive functions in both sexes of rats, but the response in females is greater than in males.
While aldosterone receptor blockers improve survival of patients with congestive heart failure, spironolactone and its derivatives were recently shown to block ether-a-go-go-related gene (HERG) channels and native IKs and IKr currents in guinea pig ventricular myocytes. In this study, we examined in vivo electropharmacological effects of an active derivative of spironolactone, potassium canrenoate, using a halothane-anesthetized canine model. Potassium canrenoate was intravenously administered in three doses of 1, 10, and 100 mg/kg per 10 min with a pause of 20 min between doses (n = 5). The low dose hardly affected any of the cardiovascular parameters. The middle dose, a clinically recommended daily maximum i.v. dose, slightly inhibited the intraventricular conduction. The high dose decreased the heart rate, ventricular contraction and blood pressure, delayed the atrioventricular and intraventricular conduction, and prolonged the ventricular repolarization and refractory period. Increment in the refractoriness by the high dose was greater than that in the repolarization, resulting in the reduction of ventricular electrical vulnerability. This unique electrophysiological profile of potassium canrenoate may in part contribute to the favorable clinical results, whereas caution has to be paid on the cardiohemodynamic actions, particularly for patients with risk of elevated plasma drug concentration.
The clinical effectiveness of gabapentin for the treatment of epilepsy, spasticity, and neuropathic pain has been established. The mechanisms responsible for those actions, however, are still not clearly understood. We have recently demonstrated that gabapentin reduces the spinal reflex in rats via mechanisms that do not involve γ-aminobutyric acid (GABA)A receptors. In the study, we attempted to explore the involvement of GABAB receptors in gabapentin-induced inhibition of the spinal reflexes in spinalized rats. Stimulation of the dorsal root at L5 elicited the segmental mono-(MSR) and polysynaptic reflex (PSR) in the ipsilateral ventral root. The microinjection of gabapentin (1.5 and 5 nmol) into the ventral horn reduced both MSR and PSR, whereas the injection into the dorsal horn only inhibited the PSR, indicating that systemic gabapentin inhibits the MSR at the ventral horn and it inhibits the PSR at both the ventral and dorsal horns. The GABAB-receptor antagonist CGP35348 (0.5 nmol) injected into the ventral horn antagonized the inhibition of the spinal reflexes by the GABAB-receptor agonist baclofen (i.v.) but not by gabapentin (i.v.). Thus, GABAB receptors do not appear to contribute to the gabapentin-induced inhibition of the spinal reflex.
Simultaneous administration of psychostimulants and opioids is a major drug abuse problem worldwide. This combination appears to produce synergistic effects on behavior at low doses; however, there is little direct evidence that the combination is stronger than either drug alone. Therefore, we investigated interactions between psychostimulants and morphine on locomotor activity in mice. Low doses of cocaine (5.0 mg/kg) or methamphetamine (0.5 mg/kg) and morphine (10 mg/kg) enhance locomotor activity in a synergistic fashion. Effective doses of cocaine (20 mg/kg) and morphine (20 mg/kg) increased locomotion in an additive fashion. In contrast, combination of methamphetamine (1.0 and 2.0 mg/kg) and morphine (10 and 20 mg/kg) did not merely enhance their effects (or attenuated the peak effects of methamphetamine-induced hyperlocomotion). These results indicate that different mechanisms explain the interaction between morphine and methamphetamine or cocaine. It is well known that psychostimulants- and opioids-induced hyperlocomotion is mediated by the activation of the dopaminergic system, however, haloperidol (a dopamine receptor antagonist) and U50,488H (which attenuates dopamine release from nerve terminals) significantly increased the effects of methamphetamine and morphine on the locomotor activity. These results suggest that excess dopaminergic activation may be involved in the effects of methamphetamine and morphine on locomotor activity in mice.
Methamphetamine induces hyperlocomotion, and high doses of methamphetamine induce self-injurious behavior (SIB) in rodents. It is well known that the monoaminergic system is involved in methamphetamine-induced behavior. However, the effects of dopamine- and serotonin (5-HT)-related compounds on high-dose methamphetamine-induced behavior have not been sufficiently clarified. Therefore, the present study was designed to investigate the effects of dopamine receptor antagonists and indirect 5-HT receptor agonists on high-dose methamphetamine-induced behavior in mice. Methamphetamine (20 mg/kg) initially increased locomotor activity. As the dosage increased, continuous SIB accompanied by a reduction in locomotor activity was observed. The hyperlocomotion and SIB induced by 20 mg/kg of methamphetamine was abolished by high doses of SCH23390 and haloperidol, indicating that the hyperlocomotion and SIB induced by high doses of methamphetamine are mediated by the activation of D1- and D2-receptors. Furthermore, haloperidol (0.1 mg/kg) potently increased locomotor activity in combination with 20 mg/kg methamphetamine. These results suggest that excess dopaminergic activation, especially activation of dopamine D2-receptors, may be involved in the decrease in locomotor activity induced by a high dose of methamphetamine. On the other hand, indirect 5-HT receptor agonists attenuated methamphetamine-induced SIB, suggesting that the stimulation of 5-HT receptors plays an important role in high-dose methamphetamine-induced SIB in mice.
Epileptic patients exhibited variably altered status of trace elements, electrolytes, and free radical scavenging enzyme activities. We investigated the effect of epilepsy and long-term antiepileptic drug therapy on the serum level of some trace elements (zinc, selenium, and copper), electrolytes (calcium, magnesium, sodium, and potassium), and antioxidants (glutathione peroxidase, and uric acid) and plasma levels of lipid peroxidation index (malondialdehyde), total antioxidant capacity, and ceruloplasmin. Seventy epileptic patients and fourteen controls were recruited in this study. In the treated group (particularly with valproate), we reported increases in the levels of zinc, calcium, sodium, malondialdehyde, and glutathione peroxidase and decreases in the levels of copper, total antioxidant capacity, and ceruloplasmin with no difference in selenium, magnesium, and potassium. However among untreated epileptics, uric acid level was increased and total antioxidant capacity was markedly lowered. We conclude that the above parameters balance differs in epileptics comparable to controls and hence their correlation to seizures pathophysiology and their degree of control or resistance to antiepileptic drug therapy. Better regulation of the lipid peroxidation and antioxidants and fewer disturbances in mineral metabolism were observed in monotherapy versus polytherapy and with carbamazepine versus valproate therapy.
Homologous and heterologous phosphorylations of histamine H1 receptor (H1R) in intact cells were investigated using Chinese hamster ovary cells stably co-expressing c-myc-tagged human histamine H1 and muscarinic M3 receptors. Increase in histamine-induced homologous phosphorylation of H1R was induced in a dose- and time-dependent manner. Maximum phosphorylation of H1R by 8-fold over the basal level was induced 1 min after the stimulation, and the increased phosphorylation level was maintained over 40 min. M3 receptor-mediated heterologous phosphorylation of H1R reached maximum by 2-fold over the basal level at 5 min after the stimulation and then rapidly returned to the basal level by 40 min after the stimulation. Histamine-induced phosphorylation of H1R was partially inhibited by three protein kinase inhibitors including Ro-31-8220 for protein kinase C (PKC), KN-93 for calcium/calmodulin-dependent kinase II (CaMKII), and KT5823 for protein kinase G (PKG), while, M3-receptor-mediated phosphorylation of H1R was completely inhibited by Ro 31-8220. Protein kinase activators including phorbol 12-myristate 13-acetate (PMA), 8-bromo-cyclic GMP (8-Br-cGMP), and 8-bromo-cyclic AMP (8-Br-cAMP) induced increases in H1R phosphorylation. Increased phosphorylation of H1R, by 5-fold over the basal level, induced with a combination of PMA, 8-Br-cGMP, and 8-Br-cAMP was still lower than that with histamine. It was suggested that H1R-mediated H1R phosphorylation involves the activation of PKC, CaMKII, PKG, and other unidentified kinases including G-protein coupled receptor kinases (GRKs) and that PKC is solely involved in M3 receptor-mediated H1R phosphorylation.
We examined the role of interstitial cells of Cajal (ICC) in the ascending and descending neural reflexes in the ileal segments prepared from wild type mice and c-kit mutant W/WV mice. Localized distension of the ileal segments from wild type mice with a small balloon caused contraction or relaxation of the circular muscle on the oral or anal side of the distended region, respectively. However, these intestinal reflexes were not induced in the ileal segments from the mutant mice. In the small strips that include the step of the pathways from efferent motor neurons to smooth muscle cells, nerve stimulation induced contraction of circular muscle in the absence of atropine and relaxation in the presence of atropine. The extent of nerve stimulation-induced contractions and relaxations of the ileal circular muscle were similar in wild type and W/WV mice. The responsiveness of ileal circular muscle to exogenously added acetylcholine and Nor-1, a nitric oxide donor, was also unaffected in the mutant ileum. Since previous immunohistochemical study had revealed selective loss of ICC within the myenteric plexus (ICC-MY) in the mutant ileum, it was concluded that ICC-MY have an essential role in ascending and descending neural pathways in the mouse ileum.
The role of extracellular calcium in the interaction between intracellular cAMP and nitric oxide (NO)/cGMP on the contractility of rat diaphragm pretreated with cumulative concentrations of aminophylline (0.36 – 3.60 mM) was investigated. In a Ca2+-free medium, NG-nitro-L-arginine methyl ester (L-NAME) (1 and 3 mM) depressed tension developed (Td) and also aminophylline-induced potentiation of Td in a concentration-dependent manner. Verapamil (2.5 μM) or nicardipine (20 μM) significantly antagonized the potentiating effect of L-NAME on Td in a calcium-containing medium. However, in the presence of verapamil or nicardipine, L-NAME still produced statistically significant potentiation of the cumulative concentrations of aminophylline (0.36 – 3.60 mM), given in the second series.
In guinea pig ventricular cardiomyocytes, the R(−)-enantiomer of efonidipine concentration-dependently blocked T-type Ca2+ current with 85% inhibition at 1 μM. In contrast, R(−)-efonidipine (1 μM) had no effect on the L-type Ca2+ current and Ca2+ transient in cardiomyocytes and contractile force in papillary muscles. Thus, R(−)-efonidipine is a highly selective blocker of the T-type Ca2+ current in native myocardia.