Up-regulation of calcitonin gene-related peptide receptors underlying elevation of skin temperature in ovariectomized rats.

We investigated the mechanism for the augmentation of the calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature in ovariectomized (OVX) rats. I.v. injection of alphaCGRP (10 micro g/kg) elevated skin temperature of the hind paws. The elevation was significantly greater in OVX rats than in sham-operated rats and was inhibited by pretreatment with human CGRP(8-37) (100-1000 micro g/kg i.v.), a CGRP receptor antagonist, in a dose-dependent manner. In addition, ovariectomy not only potentiated vasorelaxation due to alphaCGRP but increased the number of CGRP receptors in mesenteric arteries. Further, the plasma concentration of endogenous CGRP was significantly lower in OVX rats. These results suggest that the low concentration of plasma CGRP due to ovarian hormone deficiency may induce the increase in the number of CGRP receptors due to up-regulation. Therefore, the increased number of CGRP receptors may be responsible for potentiation of exogenous alphaCGRP-induced elevation of skin temperature in OVX rats. The mechanism underlying the hot flashes observed in menopausal women may also involve, in part, the up-regulation of CGRP receptors following ovarian hormone deficiency.

Effects of the Japanese herbal medicine Keishi-bukuryo-gan and 17beta-estradiol on calcitonin gene-related peptide-induced elevation of skin temperature in ovariectomized rats.

The effects of a Japanese herbal medicine, Keishi-bukuryo-gan, and 17beta-estradiol on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in ovariectomized (OVX) rats. Ovariectomy not only potentiated CGRP-induced elevation of skin temperature and arterial vasorelaxation but also induced a lower concentration of endogenous CGRP in plasma and up-regulation of arterial CGRP receptors, suggesting that lowered CGRP in plasma due to ovarian hormone deficiency increases the number of CGRP receptors and consequently amplifies the stimulatory effects of CGRP to elevate skin temperature. Oral Keishi-bukuryo-gan (100-1000 mg/kg, once a day for 7 days) restored a series of CGRP-related responses observed in OVX rats by normalizing plasma CGRP levels in a dose-dependent manner as effectively as s.c. injection. 17Beta-estradiol (0.010 mg/kg, once a day for 7 days). However, Keishi-bukuryo-gan did not affect the lower concentration of plasma estradiol and the decreased uterine weight due to ovariectomy, although the hormone replacement of 17beta-estradiol restored them. These results suggest that Keishi-bukuryo-gan, which does not confer estrogen activity on plasma, may be useful for the treatment of hot flashes in patients for whom estrogen replacement therapy is contraindicated, as well as menopausal women.

Chronic stress differentially regulates glucocorticoid negative feedback response in rats.

Exposure to chronic stress is thought to play an important role in the etiology of depression. In this disorder, a disrupted negative feedback response to exogenous glucocorticoids on cortisol secretion has been indicated. However, the regulation of glucocorticoid negative feedback by chronic stress is not fully understood. In the present study, we investigated the effects of chronic stress administered by water immersion and restraint (2 h/day) for four weeks on the glucocorticoid feedback in rats. In the acutely (one-time) stressed rats, the basal plasma corticosterone (CORT) level was markedly elevated, remained at high levels for 5 h after the termination of stress, and then decreased. In the chronically stressed rats, the CORT level was initially elevated similarly, but rapidly decreased at 2 h. In the dexamethasone (DEX) suppression test, the peak CORT level in response to stress was not suppressed by DEX in the acutely stressed rats, but was significantly suppressed in the chronically stressed rats. In contrast, the suppressive effects of DEX on the basal CORT secretion in naive rats were attenuated in the chronically stressed rats. In the chronically stressed hippocampus, which plays an important role in the regulation of the glucocorticoid feedback response, the binding of [3H]DEX was decreased and the increased response of activator protein-1 induced by acute stress was abolished. These results suggest that chronic stress induces a hypersuppressive state for induced CORT secretion in response to acute stress, which is caused by partial habituation, coping, and adaptation to the stressor, whereas it induces a hyposuppressive state for the basal CORT secretion, which is caused by glucocorticoid receptor downregulation. These mechanisms may be involved in the stress-induced neural abnormalities observed in depression.

Effects of the vasoactive neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal polypeptide on skin temperature in ovariectomized rats.

The effects of three vasoactive neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal polypeptide (VIP), on vasodilation and skin temperature were investigated in ovariectomized (OVX) and sham-operated control rats. CGRP (0.01-1 nmol), VIP (0.01-10 nmol) and SP (0.1-100 nmol) produced vasodilation in PGF(2 alpha) (10 microM)-induced contraction of mesenteric vascular beds isolated from OVX and sham-operated rats in a dose-dependent manner. Intravenous injection of CGRP (1-10 microg/kg), VIP (10-50 microg/kg) and SP (10-50 microg/kg) elevated the skin temperature in OVX and sham-operated rats in a dose-dependent manner. CGRP had the greatest effect on both parameters, followed by VIP, with the smallest effect in SP. These parallel increases of vasodilation and skin temperature with CGRP were significantly greater in OVX rats than in sham-operated rats. However, no significant differences were observed in VIP- or SP-induced vasodilation and skin temperature increases between OVX and sham-operated rats. These results suggest not only that CGRP is closely related to the elevation of skin temperature but also that CGRP-induced responses are more affected by ovarian hormone deficiency.

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum.

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABAA and GABAB receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABAA receptor binding without significantly altering GABAB receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABAA, but not GABAB receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABAA receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABAB receptors located on non-cholinergic neuronal elements in the striatum.

Effect of chronic stress on cholinergic transmission in rat hippocampus.

We previously demonstrated that chronic stress impaired prefrontal cortex-sensitive working memory, but not reference memory. Since the hippocampal cholinergic system is also involved in these memories, we examined the effects of chronic stress on cholinergic transmission in the rat hippocampus. A microdialysis study revealed that the stress did not affect the basal acetylcholine release, but enhanced the KCl-evoked response. These results suggest that cholinergic transmission in the chronically stressed hippocampus does not contribute to working memory impairment, but it may be involved in maintenance of reference memory.

Damage of hippocampal neurons caused by cobalt focus in the cerebral cortex of rats.

The relationship between the focus of cobalt seizures in the cerebral cortex and neuron loss in the hippocampus, as well as the CD50 of pentylenetetrazol was examined in rats. Spike activity in EEG frequently appeared 4 days after cobalt application and reached a peak 8-16 days after cobalt application, which was sometimes accompanied by jerks in the limbs. Changes in the CD50 value showed a two-step pattern, i.e., the first decrease in CD50 appeared one day after application of cobalt and continued at the same value until the fourth day. Then a second gradual decrease of CD50 was observed from the fourth day to eighth day and continued at the same value until 20 days after cobalt application. Neuron loss in the CA1 area of the hippocampus was observed as early as two days after cobalt application and the degree of neuron loss progressively increased until the 20th day. These findings suggest that neuron loss in the hippocampus following cobalt-induced seizures is not a result of generalized convulsions.

Direct regulation of acetylcholine release by N-methyl-D-aspartic acid receptors in rat striatum.

The aziridinium ion of ethylcholine (AF64A), a cholinergic neurotoxin, was injected into the right striatum of a rat. The unilateral injection of 10 nmol AF64A reduced the activity of choline acetyltransferase (CAT) and the tissue content of acetylcholine (ACh) in the striatum. The striatal contents of dopamine (DA), norepinephrine (NE), 5-hydroxyindoleacetic acid (5-HIAA) and gamma-aminobutyric acid (GABA) were unchanged. These results suggest that the cholinospecificity in the striatal lesion was induced by the 10 nmol dose of AF64A. The number of N-methyl-D-aspartic acid (NMDA) receptors in the striatum treated with 10 nmol AF64A was determined by a specific binding assay using [3H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), a selective ligand for NMDA receptors. The number of the NMDA receptors decreased significantly in the injected area. On the other hand, in a microdialysis using normal rats, the perfusion of 50 microM NMDA into the striatum increased ACh release. The perfusion of 100 microM MK801 which is the specific and non-competitive NMDA receptor antagonist, decreased the basal levels of ACh release and blocked NMDA-elicited ACh release. Taken together, the present results strongly suggest that a population of NMDA receptors exists on cholinergic interneurons within the striatum, and it directly regulates ACh release.

An inhibitory effect of cytokine-induced neutrophil chemoattractant on corticotropin-releasing factor-induced increase in locomotor activity.

To investigate whether cytokine-induced neutrophil chemoattractant (CINC) has an influence on corticotropin-releasing factor (CRF) in the central nervous system, the effects of intracerebroventricular (i.c.v.) injection of CINC on CRF-induced behavior were examined. Intracerebroventricular CRF injection produced an increase in locomotor activity, which was significantly reduced by i.c.v. injection of CINC. The intravenous injection of CINC did not alter CRF-induced locomotor hyperactivity. These results suggested that CINC has a functional antagonistic action on the response to CRF and may attenuate stress responses.

Effects of cycloheximide on delayed neuronal death in rat hippocampus.

The effect of cycloheximide, a protein synthesis inhibitor, on hippocampal selective neuronal death was morphologically studied in rats subjected to 10 min forebrain ischemia using a 4-vessel occlusion model. Neuronal damage in the hippocampal CA1 subfield 72 h after ischemic insult was dramatically decreased by the lasting inhibition of protein synthesis through consecutive administration of cycloheximide. Cycloheximide, which was administered once within the first 24 h of recirculation, showed protective action on ischemic cell necrosis and its most potent effect was observed when injected at 12 h of post-ischemia. After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent cell death. The possibility is considered that hippocampal delayed neuronal death following transient ischemia is caused by abnormal protein(s).

Neurochemical determination of the location of NMDA and GABA receptors on rat striatal cholinergic neurons.

This paper reports on the protocol for neurochemical determination of the location of various receptors on cholinergic neurons in various brain regions. We applied this protocol to investigate whether NMDA and GABA receptors are located on rat striatal cholinergic neurons. When striatal cholinergic neurons were selectively destroyed by intrastriatal injection of cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64A), the number of NMDA and GABA(A) receptors decreased. However, no significant changes were observed on the number of GABA(B) receptors. These results suggest that NMDA and GABA(A), but not GABA(B) receptors are located on cholinergic neurons in the striatum. These results also indicate the usefulness and scientific applicability of the present protocol.

Inhibitory effect of a mixture of herbal drugs (TJ-960, SK) on pentylenetetrazol-induced convulsions in E1 mice.

TJ-960 is a spray-dried mixture of 9 herbal drugs. The convulsions of E1 mice induced by pentylenetetrazol (18 mg/kg) were completely inhibited by p.o. administration of TJ-960 at a daily dose of 1.0 g/kg both in 8-week-old and 4-week-old E1 mice. These findings suggest that TJ-960 has an inhibitory effect on the convulsions of this hereditary animal model of epilepsy.

Identification of the proximal neck of giant paraclinoidal aneurysms. Technical note.

Introduction of a double-lumen Swan-Ganz balloon catheter into the parent artery facilitated precise identification of the proximal neck in two giant paraclinoidal aneurysms. Reduction of the flow rate as low as possible and positioning of the patient's head were the most important factors in this procedure.

Anxiolytic-like effect of saiboku-to, an oriental herbal medicine, on histaminergics-induced anxiety in mice.

Effect of saiboku-to, an oriental herbal medicine, on anxiety in mice was investigated using a light/dark test. Anxiogenic- and anxiolytic-like effects were evaluated on the basis of shortened and prolonged time spent in the light zone of the test. Subacute administration (once a day for 7 days) of saiboku-to (0.5-2.0 g/kg, p.o.) induced anxiolytic-like effect. To assess the effect of saiboku-to on brain histaminergic system in a state of anxiety, Compound 48/80 (1.0 microg/2 microl, i.c.v.), a non-neuronal mast cell histamine releaser, or thioperamide (10.0 mg/kg, i.p.), a neuronal histamine releaser possessing the inhibitory effect of histamine H(3) autoreceptors, induced decrease in the time spent in the light zone by co-injection with cimetidine (10.0 microg/2 microl, i.c.v.), a H(2) inhibitor, suggesting anxiety-like effect. These histaminergics-induced experimental anxieties were inhibited by pre-treatment with subacute administration of saiboku-to, as well as single treatment with diazepam. The results suggest that saiboku-to exhibits anxiolytic-like effect closely related to histaminergic system in the brain.

Effects of drugs acting as histamine releasers or histamine receptor blockers on an experimental anxiety model in mice.

Experimental anxiety in mice was evaluated using a light/dark test at 60 min after injection of various histaminergics. Thioperamide, a histamine H(3) receptor inhibitor (5-20 mg/kg, intraperitoneal [IP]), Compound 48/80, a mast cell degranulator (0.1-10 microg/2 microl, intracerebroventricularly [ICV]), mepyramine, a histamine H(1) receptor antagonist (0.1-10 microg/2 microl, ICV) or cimetidine, a histamine H(2) receptor antagonist (0.1-10 microg/2 microl, ICV) alone did not affect the locomotive activity, the time spent in the light zone, and number of shuttle crossings in the light/dark test. However, the time spent in the light zone and the number of shuttle crossings significantly decreased only when cimetidine (0.1-10 microg/2 microl, ICV) was co-treated with either thioperamide (10 mg/10 ml/kg, IP) or Compound 48/80 (1.0 microg/2 microl, ICV). The decrease in these behavioral parameters suggests induced experimental anxiety in mice. The experimental anxiety was antagonized by mepyramine (10 microg/2 microl, ICV). These results suggest that not only neuronal histamine release induced by thioperamide but also non-neuronal (mast cells) histamine release induced by Compound 48/80 play an important role in inducing experimental anxiety via post-synaptic H(1) and H(2) receptors. In addition, it is likely that the anxiety may be mediated by the stimulation of H(1) receptors, while H(2) receptors may inhibit the anxiety produced by the activation of H(1) receptors.

Interaction of drugs and Chinese herbs: pharmacokinetic changes of tolbutamide and diazepam caused by extract of Angelica dahurica.

The inhibitory effects of Angelica dahurica root extract on rat liver microsomal cytochrome P450 and drug-drug interactions were studied. The 2alpha- and 16alpha-hydroxylase activity of testosterone were most strongly inhibited, with 17.2% and 28-5% of their activity remaining, respectively, after oral administration of A. dahurica extract at a 1 g kg(-1) dose. 6beta-Hydroxylase activity was also inhibited, with 70% of its activity remaining, under the same conditions. In addition, treatment with the extract inhibited the metabolism of tolbutamide, nifedipine and bufuralol. These results showed that the extract inhibited the various isoforms of cytochrome P450 such as CYP2C, CYP3A and CYP2D1. The A. dahurica extract delayed elimination of tolbutamide after intravenous administration at a 10 mg kg(-1) dose to rats. Thus, the extract altered the liver intrinsic clearance. It had little effect, however, on the pharmacokinetic parameters of diazepam after intravenous administration at 10 mg kg(-1). Since diazepam showed high clearance, it underwent hepatic blood flow rate-limited metabolism. Therefore, the change of intrinsic clearance had little effect on hepatic clearance. However, the Cmax value after oral administration of diazepam with extract treatment was four times that with non-treatment. It was suggested that the first-pass effect was changed markedly by the extract. High-dose (1 g kg(-1)), but not low dose (0.3 g kg(-1)), administration of A. dahurica extract increased significantly the duration of rotarod disruption following intravenous administration of diazepam at 5 mg kg(-1). It was concluded that administration of A. dahurica extract has the potential to interfere with the metabolism, by liver cytochrome P450, of other drugs.

Effect of Saiboku-to, an Oriental Herbal Medicine, on gastric lesion induced by restraint water-immersion stress or by ethanol treatment.

The effect of saiboku-to on gastric lesions induced by restraint water-immersion stress and ethanol has been examined in rats. Thirty minutes after oral administration of saiboku-to, the rats were placed in restraint cages and immersed in water at 23 degrees C for 7 h, or orally administered 99.5% ethanol (1 mL) and placed in normal cages for 1 h. The stress for 7 h or the ethanol treatment for 1h induced erosion in the glandular area of the stomach. Histology showed that the surface epithelial cells were desquamated and part of the lamina propria mucosae was injured. The evaluation of lesion index, the cumulative length of the gastric lesion, on the gross appearance of the stomach, revealed that saiboku-to dose-dependently inhibited both the water-immersion stress-induced gastric erosion and ethanol-induced gastric erosion. To determine whether the anti-erosion effect of saiboku-to was because of a mild irritant effect, saiboku-to or 20% ethanol, which is known as a typical mild irritant, were given orally. After 30 min a strong irritant, 99.5% ethanol, was given orally. Histological examination was performed 30 min after administration of saiboku-to or the mild irritant, and 1 h after administration of the strong irritant. The mild irritant induced a reduction in surface epithelial cells 30 min after administration. Furthermore, the mild irritant protected the stomach against mucosal erosion produced by the strong irritant. Saiboku-to protected the strong irritant-induced erosion without producing mild irritation as observed in stomach treated with 20% ethanol. Pretreatment with saiboku-to also inhibited the decrease in the levels of hexosamine, gastric mucus glycoprotein, induced by the strong irritant. In pylorus-ligated rats, saiboku-to dose-dependently inhibited gastric acid secretion, a gastric aggressive factor. These results suggest that the anti-erosion effect of saiboku-to which is not a mild irritant, involves both inhibition of aggressive factors, such as gastric acid secretion, and augmentation of defensive factors, such as gastric mucus cells.

Proliferative effect of ginseng saponin on neurite extension of primary cultured neurons of the rat cerebral cortex.

The effect of the crude saponins of Panax ginseng root on neurite extension of primary cultured neurons of the rat cerebral cortex was examined. Addition of the crude saponin extract to the culture medium showed a proliferative effect of neurite extension in 24-h and 48-h cultures. The looping phenomenon and disappearance of the growth cone due to the addition of cytochalasin-B to the culture medium were protected by the addition of the crude saponin. Addition of the crude saponin extract showed increases of about 1.5-fold in ganglioside content of cultured neurons. These results suggest that the crude saponin extract of the ginseng root has a promoting effect on neurite extension and also a protecting effect on distortion of neurites due to cytochalasin-B.

Pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine without effects on histamine H1 receptors and muscarinic cholinergic system in the brain.

The pharmacological characteristics of Sho-seiryu-to, an antiallergic Kampo medicine, were investigated. Forty-eight-hour passive cutaneous anaphylactic (PCA) reaction was significantly inhibited in rats orally administered Sho-seiryu-to (1000 mg/kg). Sho-seiryu-to significantly inhibited increase in vascular permeability induced by histamine. These data confirm previous findings that Sho-seiryu-to has antiallergic activity in animals and suggest that the antagonism of histamine may be an antiallergic mechanism of Sho-seiryu-to. Sho-seiryu-to did not affect locomotor activity or motor coordination in mice. Although ketotifen prolonged sleeping time induced by pentobarbital, Sho-seiryu-to had no such effect. Nor was there any effect on oxotremorine-induced tremor and [3H]-mepyramine binding to histamine H1 receptors in rat brain. Thus, Sho-seiryu-to appears to be useful for treating type I allergy, with relatively few side effects such as sedation and drowsiness due mainly to blockade of histamine H1 and muscarinic receptors in the brain.

Further pharmacological study on Sho-seiryu-to as an antiallergic.

Examination was made of the pharmacological characteristics of Sho-seiryu-to, an antiallergic kampo medicine. Sho-seiryu-to suppressed histamine release from rat peritoneal mast cells, but failed to inhibit the binding of [3H]-mepyramine to histamine H1 receptors in guinea pig cerebral cortex and lung. Sho-seiryu-to had no effect on cutaneous reactions induced by serotonin, platelet-activating factor (PAF), leukotriene (LT) C4 or LTD4. Ketotifen prolonged electrically induced convulsions, while Sho-seiryu-to did not. Sho-seiryu-to did not affect salivation induced by pilocarpine. Sho-seiryu-to thus does not appear to inhibit histamine H1 receptors or inflammation induced by serotonin, PAF, LTC4 and LTD4, but suppresses mast cell activity. Sho-seiryu-to would thus have only a few side effects such as dry mouth and convulsions due mainly to the blockage of the action of muscarinic in salivary glands and histamine in the brain.