Involvement of glucocorticoid receptor on hyperpyrexia induced by methamphetamine administration.

UNLABELLED: We have investigated the involvement of glucocorticoid on methamphetamine (MA) induced hyperpyrexia using a bio-telemetric system. A significant level of hyperpyrexia was observed in MA administered rats. In contrast, increase of body temperature was suppressed by adrenalectomy or by the administration of RU-486, an antagonist of the glucocorticoid receptor. These data suggest that the glucocorticoid receptor may be involved in hyperpyrexia induced by MA. KEYWORDS: methamphetamine - hyperpyrexia - glucocorticoid - corticosterone.

Cholinergic nerves mediate acetaldehyde action in the gastrointestinal tract.

The regulation mechanism of inhibition of intestinal ethanol absorption induced by high acetaldehyde (AcH) concentration in blood was investigated. We used atropine (AT), atropine methylbromide (ATMB), pirenzepine (PI), bethanechol (BE) and pilocarpine (PL) with or without cyanamide (CY; a potent inhibitor of aldehyde dehydrogenase, which induces high AcH concentration in blood). The K(a) (absorption rate constant) value after the CY-alone pretreatment was significantly lower than that in controls. In the high AcH-induced cases, the values of K(a) in AT and ATMB pretreatments were similar to controls, but the value of K(a) in PI pretreatment was lower than that in controls. The values of K(a) in the case of BE pretreatment with and without high AcH levels were lower than in controls. The K(a) value in the PL with CY was significantly lower than that with CY alone. However, its action was blocked by ATMB pretreatment. These results suggest that high blood AcH concentrations inhibit intestinal ethanol absorption through the peripheral cholinergic nerves via muscarinic receptors, except for the muscarinic M(1) receptor, compared to other subtypes of muscarinic receptors.

[The false positive reaction of the Triage panel drug-of-abuse by herbal drugs ma-huang (Ephedra sinica (Ephedraceae))].

We investigated false-positive reactions obtained from a drug screening test using a Triage panel. We detected 2 cases giving false-positive reaction for AMP (amphetamine, methamphetamine) during the screening of 187 normal subjects. Subsequent follow up testing by high-performance liquid chromatography (HPLC), showed both to be false-positive reactions. As both cases have a history of ingesting the herbal drug, Ma-huang (Ephedra sinica (Ephedraceae)), containing ephedrine, we examined the relationship between false-positive reactions on Triage and Ma-huang. All urine samples collected from 7 healthy volunteers following administration of Ma-huang indicated AMP positive on Triage. Also a high ratio of AMP positives was observed in the patients who were administered Ma-huang-containing drugs at the hospital. However, none of them were identified as true-positives by HPLC or gas chromatography mass spectrometry (GC/MS) analysis. The extract of Ma-huang contained in herbal drugs, which otherwise contain neither amphetamine nor its derivatives, gives (AMP) positive indications on Triage. We speculate that unidentified components of Ma-huang cause the false-positive reactions. We suggest that follow-up tests by GC/MS or HPLC are needed wherever a positive result is obtained from a screening test by Triage. Furthermore, it will be established to continue collecting information on prescribed and non-prescribed drugs.

Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor.

A model of atrophic rat submandibular gland was used to examine the ability of basic fibroblast growth factor (bFGF) to accelerate tissue repair. The gland duct was separated carefully from associated blood vessels and nerve, and ligated with a 8-0 suture under a surgical microscope. Two weeks after ligation, the glandular tissue showed severe atrophy and weight loss (to 26% of that in a sham-operated group). Thereafter, the ligature was removed and various amounts of bFGF, isoproterenol or saline were instilled retrogradely through the duct. Both isoproterenol and bFGF increased cell proliferation significantly. bFGF accelerated the proliferation of various cell types, including both acinar and ductal. The proliferative effects of bFGF peaked at a dose of 1 ng/gland. When bFGF (1 ng/gland) was administered to the atrophic gland, its weight increased to 125% of the glands in saline-treated control animals after 2 weeks. The effects of bFGF were also examined in normal submandibular glands: bFGF stimulated cell proliferation, but the effective concentration was at least 50 times higher than that required in the atrophic gland. The results from immunohistochemical tests against anti-FGF receptor-type 1 antibody demonstrated increased immunoreactivity in the damaged gland, which might be involved in the difference in the response to bFGF between damaged and normal glands. Overall, the results indicate that bFGF can accelerate tissue repair in salivary gland.

[Simultaneous administration of ethanol emphasizes the effect of methamphetamine on central nervous system in rat with high alcohol preference].

We studied about influence of alcohol preference and of simultaneously administered ethanol (EtOH) on the effect of methamphetamine (MA) on the central nervous system neurochemically and behavioral pharmacologically using two strains of rat with high or low preference for alcohol (HAP and LAP). In the neurochemical study, we determined dopamine (DA) and serotonin (5-HT) level in striatum and nucleus accumbens (N.Acc) by means of brain microdialysis after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group) every 20 minutes for 10 sessions. In the behavioral pharmacological study, we observed rat's behavior in the open field and performed scoring according to Locomotion-Stereotypy (L-S) rating scale by Ellinwood every five minutes, and counted locomotion and rearing by means of infrared beam cutting every 20 minutes after administration of MA 1 mg/kg alone (LAP-MA group, HAP-MA group) or EtOH 2 g/kg + MA 1 mg/kg (LAP-EtOH/MA group, HAP-EtOH/MA group). When MA was given alone, HAP rat showed no significant difference in locomotion, rearing and L-S score compared to LAP rat, although HAP rat showed significantly lower elevation of DA and 5-HT in both striatum and N.Acc. When EtOH was simultaneously administered with MA, in LAP rat, DA and 5-HT elevation in the striatum and N.Acc showed no significant differences between LAP-EtOH/MA group and LAP-MA group. Locomotion and rearing reduced and L-S score temporarily reduced significantly in LAP-EtOH/MA group compared to LAP-MA group. However in HAP rat, HAP-EtOH/MA group showed significantly higher DA and 5-HT elevation in both striatum and N.Acc, and also showed significantly higher L-S score and higher locomotion count compared to HAP-MA group. Our results indicate that effect of MA may be influenced by alcohol preference, and that simultaneous administration of EtOH emphasizes the effect of MA on central nervous system in rat with high alcohol preference.

Salivary growth factors in health and disease.

The salivary gland is considered to be a reservoir of many growth factors in rodents. In humans, the epidermal growth factor, basic fibroblast growth factor, and insulin and insulin-like growth factor family have also been detected in this gland, but their physiological role remains unclear. In this study, we focused on bFGF, which is a well-known mitogen for various types of cells, and is present in the salivary gland as well as in saliva. The roles of bFGF in the salivary gland were investigated by three different procedures. First, the effects of bFGF on the salivary gland cells were investigated with a monolayer culture of normal submandibular gland cells. The effects of different concentrations of bFGF on the second passage of these cultured cells were examined. In both human and rat cultured submandibular gland cells, bFGF accelerated the cell proliferation at a concentration of 100 ng/mL or higher. Next, an atrophic model of the rat submandibular gland was used to examine the ability of bFGF to accelerate tissue repair. Two weeks after ductal ligation, the ligature was removed, and various amounts of bFGF, isoproterenol, or saline were administered via a retrograde duct instillation. Both isoproterenol and bFGF increased acinar and ductal cell proliferation significantly. To determine the role of bFGF in saliva, we investigated its effect on the healing process of oral mucosal defects. Four-millimeter mucosal defects were made to the depth of the periosteum in the rat palate under anesthesia. bFGF or vehicle alone was applied once only at the time of surgery as a suspension. At days 3, 5, and 7 in the bFGF group, significant increases in the degree of re-epithelialization were found in treated groups. These results indicate that its action as a mitogen stimulus is the major effect of bFGF on salivary gland cells and mucosal epithelium.

Ethanol inhibits intra- and extracellular Ca(2+)-dependent contraction of rat aorta by different mechanisms.

The mechanism of the inhibitory action of ethanol on vasoconstriction was studied by examining ethanol effects on vascular responses to different vasoconstrictive stimulants using isolated rat aorta. Ethanol pretreatment of aortic strips at 200 and 400 mM significantly inhibited contractile responses to KCl and 5-hydroxytryptamine in the standard medium. This inhibitory effect on vasoconstriction was observed whether or not the endothelium was present. Pretreatment with ethanol (400 mM) caused significant decrease of 45Ca2+ uptake induced by KCl when compared to the control. This pretreatment also significantly inhibited the contractile response to 5-hydroxytryptamine in the medium without Ca2+, decreased 5-hydroxytryptamine-stimulated inositol monophosphate accumulation, and significantly inhibited a transient contraction induced by 20 mM caffeine. Ethanol (400 mM) inhibited phorbol 12, 13-dibutyrate (PDBu)-induced contraction when Ca2+ was present in medium, but not when it was absent. These results suggest that ethanol acts on different sites of the signal transduction pathway of rat aortic smooth muscle, causing inhibition of transmembraneous Ca2+ influx through voltage-dependent Ca2+ channels, phosphoinositide hydrolysis and Ca(2+)-induced Ca2+ release. However, the cytoskeletal contractile apparatus may remain intact after ethanol treatment.

Liver cytosolic aldehyde dehydrogenase (ALDH1) polymorphism and its inheritance in Wistar rats.

Rats are very important experimental animal to study alcohol related problems. Liver aldehyde dehydrogenases (ALDHs) which metabolize aldehydes are reported to have several isozymes which are distributed in mitochondrial, microsomal and cytosolic fractions. However, there is discrepancy on reports concerning properties of cytosolic ALDH from normal liver. We report here the liver cytosolic ALDH (ALDH1) polymorphism and its inheritance in Wistar rats. Isoelectrophoretic focusing technique reveals the three ALDH1 phenotypes (termed AA, AC and CC) which are inherited in accordance with Mendelian fashion, indicating the existence of two alleles, ALDH1A and ALDH1C. In the range of pI 5.3 to 5.8, the AA phenotype possesses a major anodic band with pI 5.3 and CC phenotype also has a cathodic pI 5.8 band. In contrast, AC phenotype exhibits five bands with pIs of 5.3, 5.4, 5.5, 5.7 and 5.8. Intensity of these bands gradually diminishes from the main band with pI 5.5 to the both opposite ends. These findings suggest that AC phenotype as well as AA and CC types has a structure of tetramer which consist of the combination of subunit of the A and C gene products.

[Potentiating effect of ethanol on alpha 1 adrenergic receptor mediated contraction of thoracic aorta from guinea pig].

Using aortic strips isolated from guinea pigs, effects of ethanol on vascular smooth muscle tonus were studied. 50mM of ethanol significantly potentiated the contractions induced by norepinephrine and phenylephrine, but did not influence those induced by KCl and clonidine. Furthermore, ethanol significantly augmented the contraction induced by phenylephrine in the presence of verapamil, and did not have any effects on the contractions induced by A23187, Ba2+, PDBu and BAY K 8644. These results indicate that ethanol potentiates the alpha 1 adrenergic receptor-mediated contraction, depending on the extracellular calcium influx via the receptor-operated calcium channel.

[Application of operant conditioning techniques to forensic toxicology: experimental studies on alcohol and abusable drugs].

This paper describes some experiments that apply the operant conditioning techniques to forensic toxicological research. These techniques may be useful in investigating the mechanisms of action, toxic symptoms, legal competence and drug metabolism associated with substance abuse such as abuse of alcohol, psychotropic drugs, narcotics, stimulants, and organic solvents. 1) Genetic research on alcohol preference in rats. We applied operant conditioning to investigate alcohol preference in rats and constructed an apparatus for the measurement of discriminated operate responses for water or alcohol reinforcement in rat. This apparatus is a modified Skinner box with a one-lever two-liquid system. Fixed ratio-10 (FR-10) schedules of reinforcement are used to increase the work of the rat before it obtains the reinforcement. The voluntary choice of water or 10% ethanol by the rat can be assessed quantitatively by measuring the lever-pushing responses. It is an extremely useful method for measuring the real alcohol preference of rats. A rat was kept in a Skinner box overnight. The numbers of responses and reinforcement for water and ethanol and the volumes of the two liquids consumed were recorded. The ratio of ethanol reinforcement was defined as the number of ethanol reinforcement to the total number of ethanol and water reinforcement. The ratio of ethanol intake was defined as the volume of ethanol consumed to the volume of water and ethanol consumed. Ethanol consumption per g body weight was calculated from the volume of ethanol consumed by the rat. We used this apparatus to investigate alcohol preference of more than 300 Wistar Albino Rats, and divided them into a high alcohol preference (HAP) group and a low alcohol preference (LAP) group. Inbreeding between littermates was conducted in each of the HAP and LAP groups. The liver tissue of each offspring was obtained and the cytosol fraction was collected and subjected to isoelectric focusing using polyacrylamide gel with a pH range of 3.5 to 9.5. Examination of the electrophoretic patterns of the acetaldehyde dehydrogenase (ALDH) revealed polymorphism in cytosolic ALDH 1. We confirmed that the polymorphism follows Mendel's law of inheritance, and is governed by two codominant alleles. Moreover, the polymorphism of ALDH 1 observed in Wistar strain rats correlated closely with the differences in ethanol consumption behavior or alcohol preference. 2) Effects of abusable drugs on learning behavior of rat. In general, animal experiments for assessing drug effects, regardless whether they measure learning or intrinsic motor or physiological function, use behavioral indicators that produce a positive or negative reinforcement. In other words, the behavioral effect of a drug is assessed by whether it enhances the animal's behavior in a positive reinforcement or decreases its response in a negative reinforcement. However, the behavioral indicator of the animal is rarely an independent entity within the body, but is closely associated with other behavioral components. Even when a behavioral indicator is enhanced by a drug, whether the apparent increase is due to the enhancing effect of the drug or a result of decreases of other behavioral components cannot be judged. We therefore devised a multiple active/passive avoidance learning apparatus which is a device for analyzing drug effects in rats using a running-wheel. We used this device to assess the effects of a drug on the animal from three dimensions simultaneously; excitatory, inhibitory and discriminatory behaviors. From these we attempted to clarify the overall effect of the drug on high order learning behavior of animal. This method enables direct examination of the so-called "learning capability" of an animal, which cannot be elucidated by single scheule learning that is strongly influenced by behavioral characteristics such as excitation and inhibition. Therefore, a method which uses avoidance learning as indicator is the best method

Vasomotor nerves of vessels in the human optic nerve.

Aminergic and cholinergic vasomotor nerves in vessels of the human optic nerve were studied morphologically. Aminergic nerve fibers were observed by the glyoxylic acid method. Cholinergic nerve fibers were observed by light microscopy after acetylcholinesterase staining by the Karnovsky-Roots method and Tago's modified method. In the retrobulbar optic nerve behind the bulbus, aminergic and cholinergic vasomotor nerves were observed to be dense in the central retinal artery and vein and posterior ciliary arteries. A large number of vasomotor nerves were also demonstrated in vessels in the septum of the optic nerve, but they were sparse in pial vessels. Further centrally, a few vasomotor nerves were found in pial vessels of the intracanalicular and intracranial optic nerve, but few were observed in the septum of the optic nerve. At the optic chiasm they were densely distributed in pial vessels.

Endothelium-dependent relaxation resistant to NG-nitro-L-arginine in rat aorta.

Experiments were designed to determine whether cyclic GMP-independent relaxation is involved in the endothelium-dependent vascular relaxation response of rat aortic strip to acetylcholine. The relaxation response to acetylcholine in the presence of 3 x 10(-4) M NG-nitro-L-arginine was apparent when the precontraction was induced by norepinephrine at 5 x 10(-9) M or 10(-8) M. The relaxation response to acetylcholine resistant to NG-nitro-L-arginine was abolished by 10(-6) M atropine, 10 mM tetraethylammonium, or endothelium removal, but was not inhibited by 10(-5) M indomethacin, 3 x 10(-6) M oxyhemoglobin or 10(-5) M glibenclamide. The response was virtually abolished when the vascular strips had been preconstricted with 20 mM KCl. The increase in vascular cyclic GMP levels induced by 10(-5) M acetylcholine was completely abolished by 3 x 10(-4) M NG-nitro-L-arginine. These results suggest that acetylcholine-induced endothelium-dependent relaxation resistant to NG-nitro-L-arginine in rat aorta is unmasked when the precontractile force is caused by lower concentrations of norepinephrine and the relaxation is mediated by a cyclic GMP-independent mechanism, possibly an endothelium-derived hyperpolarizing factor.

Increased endothelium-dependent vascular relaxation in ethanol-fed rats.

We investigated the mechanism underlying increased relaxation of aortic strips to acetylcholine in rats chronically treated with ethanol. Rats were divided into three groups and maintained on liquid diets containing ethanol (35% of total calories) as the ethanol-fed group or an equicaloric volume of sucrose instead of ethanol as the sucrose-fed group for 10 weeks. The control group was also maintained on modified American Institute of Nutrition diet for the same period. Vascular strips of isolated rat aortas were mounted in organ chambers to record isometric tension. The endothelium-dependent relaxation responses to acetylcholine and calcium ionophore A23187 were greater in ethanol-fed rats than in control and sucrose-fed rats. However, the relaxation response to sodium nitroprusside or nifedipine did not differ among the three groups. Acetylcholine, calcium ionophore A23187, and sodium nitroprusside caused an increase in the cGMP contents of rat aortic strips that was similar among the three groups. These results suggest that a cGMP-independent relaxation mechanism is involved in the increased relaxation response to acetylcholine after chronic treatment with ethanol.

[A case of sudden death of a patient with hypopituitarism].

We report a 29-year-old woman who died due to pituitary insufficiency. After a normal delivery 2 years previously, she had suffered from amenorrhea and displayed decreased libido. She was discovered in convulsions by her husband when he returned home, and was admitted to an emergency hospital. Despite various treatments, she died. Her blood glucose level on admission was low, 32 mg/dl. Autopsy findings showed thin public hair and almost no axillary hair. The right and left adrenal glands weighed 1.7 g and 1.2 g, respectively. Histologically, the anterior pituitary gland showed severe bleeding and necrosis, and the middle lobe showed lymphocytic infiltration; the posterior lobe was almost normal. The adrenal glands showed marked atrophy of the cortex and deposition of calcium in the medulla. The thyroid gland, which weighted 15 g, showed diffuse interstitial lymphocytic infiltration, indicative of chronic lymphocytic thyroiditis. Atrophy of the ovaries and uterine endometrium was also observed. These findings indicated that death had been due to pituitary insufficiency. The histopathology of the pituitary, which showed lymphoid hypophysitis, and its association with lymphoid thyroiditis suggested that the pituitary insufficiency was not due to ischemic injury after delivery, a condition which can result from massive hemorrhage, but rather had arisen as a result of an autoimmune process.

Vasorelaxant effects of oxpentifylline and theophylline on rat isolated aorta.

The mechanism of the relaxation response of rat aorta to the phosphodiesterase inhibitors oxpentifylline and theophylline was studied. Oxpentifylline induced a greater vasorelaxation response in the intact strips than in those without endothelium. The endothelium-dependent relaxation response to oxpentifylline was inhibited by nitro-L-arginine but not by indomethacin, and the endothelium-independent relaxation response was potentiated by the combination with isoprenaline but not sodium nitroprusside. Theophylline induced a similar relaxation response in vascular strips with and without endothelium. The relaxation response to theophylline was not inhibited by indomethacin or nitro-L-arginine in intact strips, but was potentiated by combination with isoprenaline or sodium nitroprusside in the denuded strips. These results suggest that the two phosphodiesterase inhibitors oxpentifylline and theophylline induce vasorelaxation by different mechanisms. Oxpentifylline can induce both endothelium-dependent relaxation, which is probably mediated by an endothelium-derived relaxing factor, and endothelium-independent relaxation, which may be due to an inhibitory action on phosphodiesterase of vascular smooth muscle. In contrast, theophylline can induce endothelium-independent relaxation alone, without modulation by the endothelium.

[Inhibitory effect of fentanyl citrate on endothelium-dependent relaxation in rat aorta].

We studied the effect of fentanyl on the endothelium-dependent vascular responses in isolated rat aortic strips. Fentanyl depressed the endothelium-dependent relaxation induced by acetylcholine but not that induced by the calcium ionophore, A23187. Endothelium-independent relaxation in response to sodium nitroprusside (SNP), a soluble guanylate cyclase activator, was not depressed by fentanyl. On the other hand, fentanyl depressed the increase in cyclic GMP level stimulated by acetylcholine but not that stimulated by A23187 or SNP. Furthermore, fentanyl depressed the vasocontraction by acetylcholine but not that by histamine or KCl in isolated pig coronary artery strips without endothelium, suggesting that fentanyl can inhibit endothelium-independent contraction via muscarinic receptor on smooth muscle cells. These results suggest that fentanyl can inhibit endothelium-dependent vasorelaxation via endothelium-derived relaxing factor (EDRF) by acting on endothelial cells but not on smooth muscle cells. The inhibitory effect of fentanyl on the relaxation probably occurs at the level of muscarinic receptor on endothelial cells or at a site before biochemical pathways converting L-arginine to EDRF.

[A case of sudden death of a patient with glycogen storage disease type 1--delayed aftereffect of a traffic accident].

A case of sudden death of a 22-year-old man with glycogen storage disease type 1 is reported. Autopsy revealed striking hepatomegaly with round edges. The liver was yellowish and weighed 2840 g. Histologic findings showed excessive deposition of PAS-positive material in the hepatocytes and in the proximal renal tubular epithelium cells, which means the accumulation of glycogen. A mass surrounding splenic artery was seen in the hilus of the spleen. There was about 2000 ml of fresh blood and clots in the abdominal cavity due to bleeding from a ruptured mass. The mass was composed of a hematoma with connective tissue surrounding it and was a pseudoaneurysm. The mass was considered to have been formed from bleeding due to injury to a branch of a splenic artery. Consideration of these findings led to the conclusion that death was caused by loss of blood from rupture of the mass and that the mass was formed due to injury of the branch of the splenic artery in a traffic accident 2 years earlier but not the pathogenesis of glycogen storage disease.

Mechanism of inhibitory action of ethanol on endothelium-dependent relaxation in rat aorta.

Using isolated rat aortic strips, we investigated the inhibitory effect of ethanol on endothelium-dependent relaxation induced by acetylcholine, especially on that mediated by endothelium-derived relaxing factor. Ethanol depressed the relaxation induced by acetylcholine and inhibited the increase in the content of intravascular cyclic GMP induced by acetylcholine, but not that induced by sodium nitroprusside or calcimycin. Ethanol also inhibited the acetylcholine-induced relaxation resistant to nitro-L-arginine. These results suggest that ethanol can inhibit the cyclic GMP-dependent relaxation mediated by endothelium-derived relaxing factor. Furthermore, ethanol seems to depress the cyclic GMP-independent relaxation mechanism.

[Effect of ethanol on isometric tone of aorta from guinea pig].

Using aortic strips isolated from guinea pig, effects of ethanol on vascular smooth muscle tone were studied. Low concentrations (5, 50, 100 mM) of ethanol potentiated norepinephrine and serotonin-induced contractions, but not KCl-induced one. Ethanol (100 mM) potentiated calcium-induced contraction in the presence of norepinephrine, but not that in the presence of KCl. Ethanol (100 mM) showed no effects on norepinephrine-induced contraction in the calcium-free medium. Ethanol (100 mM) augmented 45Ca uptake stimulated with norepinephrine, but not that with KCl. Ethanol at the higher concentration of 600 mM by itself induced contraction, which was inhibited by trifluoperazine or in the calcium-free medium, but not by phentolamine, diphenhydramine, methysergide, indomethacin or nifedipine. Ethanol (600 mM) directly increased 45Ca uptake, which was not affected by nifedipine. These findings indicate that the low concentrations of ethanol potentiate receptor-mediated vascular smooth muscle contraction and the higher concentration of ethanol directly contracts it via facilitation of transmembranous calcium influx.