Different roles of nitric oxide synthase-1 and -2 between herpetic and postherpetic allodynia in mice.

We investigated using the mice role of nitric oxide synthase (NOS) in the spinal dorsal horn in herpetic and postherpetic pain, especially allodynia, which was induced by transdermal inoculation of the hind paw with herpes simplex virus type-1 (HSV-1). The virus inoculation induced NOS2 expression in the lumbar dorsal horn of mice with herpetic allodynia, but not postherpetic allodynia. There were no substantial alternations in the expression level of NOS1 at the herpetic and postherpetic stages. Herpetic allodynia was significantly inhibited by i.p. administration of the selective NOS2 inhibitor S-methylisothiourea, but not the selective NOS1 inhibitor 7-nitroindazole. NOS2 expression was observed around HSV-1 antigen-immunoreactive cells. On the other hand, postherpetic allodynia was significantly inhibited by i.p. administration of 7-nitroindazole, but not S-methylisothiourea. The activity of reduced nicotinamide adenine dinucleotide phosphate diaphorase, an index of NOS1 activity, significantly increased in the laminae I and II of the lumbar dorsal horn of mice with postherpetic allodynia, but not mice without postherpetic allodynia. The expression level of NOS1 mRNA in the dorsal root ganglia was similar between mice with and without postherpetic allodynia. The results suggest that herpetic and postherpetic allodynia is mediated by nitric oxide in the dorsal horn and that NOS2 and NOS1 are responsible for herpetic and postherpetic allodynia, respectively. It may be worth testing the effects of NOS2 and NOS1 inhibitors on herpetic pain and postherpetic neuralgia in human subjects, respectively.

Heparin and heparan sulfate enhancement of the inhibitory activity of plasminogen activator inhibitor type 1 toward urokinase type plasminogen activator.

To study effects of glycosaminoglycan on the interaction between two chain urokinase type plasminogen activator (tcu-PA) (EC 3.4.21.31) and plasminogen activator inhibitor type 1 (PAI-1) the second order rate constant (k1) between high molecular weight tcu-PA and active recombinant prokaryotic PAI-1 (rpPAI-1) was determined employing a continuous method using chromogenic substrate S-2444 either in the presence or absence of various kinds of glycosaminoglycans. k1 was (5.9 +/- 1.6).10(6)/mol per s in the absence of effector molecule, and following addition of heparin (1.0 U/ml) k1 was enhanced to (3.22 +/- 0.73).10(7). A significant enhancement of k1 was also obtained by heparan sulfate (1.87 +/- 0.25).10(7). Dermatan sulfate or chondroitin sulfate did not show a significant effect on k1 although a slight decrease was obtained by mono-dextran sulfate (4.2 +/- 1.2).10(6). The intrinsic fluorescence of rpPAI-1 was shown to be slightly increased following addition of heparin (1.49 +/- 0.22%, n = 6), suggesting that heparin may enhance the inhibitory activity of PAI-1 toward tcu-PA both by a template mechanism and by a modification of PAI-1 structure.

The significant enhancement of fibrinolysis by calcium ion in a cell free system: the shortening of euglobulin clot lysis time by calcium ion.

We investigated the roles of calcium ion (Ca2+) on euglobulin clot lysis time (ECLT) and found that the physiological concentration of Ca2+ significantly (4-5 times) shortened ECLT. The shortening was observed at the concentration of Ca2+ higher than 1.5-2.0 mM. Other divalent cations such as Mg2+, Zn2+ or Mn2+ didn't change ECLT. Anti-tPA antibody or plasminogen activator inhibitor-1 prolonged ECLT in the absence of Ca2+, whereas they had no effect on ECLT shortened by Ca2+. C1 inactivator also had no effect. When barium absorbed plasma was employed, the shortening of ECLT by Ca2+ wasn't observed, whereas it was recovered by the readdition of barium absorbed fraction. When factor X deficient plasma was employed, the shortening of ECLT by Ca2+ was also not observed. Thus, Ca2+ enhances fibrinolysis in a cell free system by a novel pathway in which the presence of factor X is prerequisite.

On the nature of three forms of phosphoenolpyruvate carboxykinase occurring in the cytosol of chicken liver.

Chicken liver mitochondria contained two forms of phosphoenolpyruvate (PEP) carboxykinase (designated as Mt-I and Mt-II) and the chicken liver cytosol fraction contained three forms of PEP carboxykinase (designated as Sol-I, Sol-II, and Sol-III). Mt-I and Mt-II were purified to homogeneity. They both had a molecular weight of 72,000 as judged by the sodium dodecyl-sulfate-polyacrylamide gel electrophoresis method, whereas an apparent molecular weight of 42,000 was obtained for both enzymes when estimated by Sephadex G-100 gel filtration. Studies with a rabbit antibody against the purified Mt-I revealed that Mt-I, Mt-II, Sol-I, and Sol-II are immunochemically identical, whereas Sol-III is immunochemically different from any of the other proteins. Genetic analogy between Mt-I and Mt-II was also suggested by gel electrophoretic analysis of their peptide maps. The content of Sol-III in the adult chicken liver was very small, whereas the livers of chick embryo and very young chick contained a considerable amount of Sol-III. The level of Sol-III in the adult chicken, however, could be significantly increased by the administration of hydrocortisone or isoproterenol. Apparently Sol-III is a cytosol-specific PEP carboxykinase which is similar to the cytosolic PEP carboxykinases of various mammals. Sol-III showed a molecular weight of about 60,000 as estimated by gel filtration. Studies with combined use of the antibody and [3H]leucine revealed that some portion of PEP carboxykinase (Sol-I plus Sol-II) appearing in the liver cytosol corresponds to a precursor in transit to the mitochondria, although major portions of Sol-I and Sol-II appear to be accounted for by release of the mitochondrial enzymes.

Some possibilities for prostaglandin mediation in the contractile response to ATP of the guinea-pig digestive tract.

Several contractile responses of longitudinal muscles of the guinea-pig digestive tract to exogenously applied ATP (10-300 micrometer), including "rebound" contractions, were inhibited by indomethacin (3-20 micrometer) or polyphloretin phosphate (10-100 microgram/ml). Relaxations to ATP in stomach and large intestinal muscles were increased by these drugs. Prostaglandin release might therefore contribute to the contractile responses of the guinea-pig digestive tract to ATP.

Impaired fibrinolytic activity induced by ingestion of butter: effect of increased plasma lipids on the fibrinolytic activity.

To investigate the effects of the increased plasma lipid level on fibrinolysis, we measured the levels of fibrinolytic components in serially obtained plasma samples from healthy volunteers after the intake of different amounts of butter. Plasma triglyceride level increased significantly after butter intake compared to the control group. Eight hours after the intake of 100g of butter, plasminogen activator inhibitor 1 (PAI-1) level in plasma was significantly higher and euglobulin clot lysis time was significantly prolonged compared to those of the control group. There was no effect on plasma tissue plasminogen activator level. These results suggest that the temporary increase in plasma triglyceride level induced high PAI-1 level, resulting in impaired fibrinolytic activity. The effect of temporary hyperlipidemia on platelet function was also analyzed and revealed that the response of platelets to ADP and collagen was lower in the butter intake group compared to those of the control.

The potential role of platelet PAl-1 in t-PA mediated clot lysis of platelet rich plasma.

The potential role of platelets in platelet rich plasma clot lysis induced by tissue plasminogen activator (t-PA) was investigated. At the various concentrations of both single chain t-PA (sct-PA) and two chain t-PA (tct-PA) (1.5nM, 3nM, and 6nM), we compared the t-PA mediated lysis time of platelet rich plasma clot (PRP-clot) with that of platelet poor plasma clot (PPP-clot). At the concentrations ranged from 1.5 to 6 nM of both types of t-PA, the clot lysis time of PRP-clot was longer than that of PPP-clot. This elongation was more significant in the tct-PA induced clot lysis than that in the sct-PA induced clot lysis. At the concentration of 3nM of tct-PA, the lysis time of PRP-clot was longer by a factor of 30% in comparison with that of PPP-clot. When the release and the aggregation of platelets were blocked by prostaglandin E1 (PGE1) and theophylline in this experiment, the lysis time of PRP-clot was essentially the same as that of PPP-clot. We then measured the antigen levels of total PAI-1 and t-PA-PAI-1 complex in the lyzed solutions of PRP-clot and PPP-clot to analyse the possible effect of plasminogen activator inhibitor-1 (PAI-1) present in platelets. Most of PAI-1 in a PPP-clot lyzed sample existed as t-PA-PAI-1 complex. In the lyzed solution of PRP-clot, however, the antigen levels of both total PAI-1 and t-PA-PAI-1 complex were significantly higher than those in PPP-clot, and larger amounts of PAI-1 existed as free PAI-1 which possesses activity. These data suggest that at least certain amounts of PAI-1 in platelets exist as an active form and inhibits t-PA activity resulting in the prolongation of the clot lysis time. Activation of platelets, therefore, seems to play an important role in the platelet rich plasma clot lysis induced by t-PA.

Electric-foot-shock induced the suppression of fibrinolytic activity in rats.

Electric-foot-shock was given to rats to initiate constant mental stress and its effect on fibrinolytic activity was analyzed. After the termination of electric-foot-shock which was given for an hour, euglobulin clot lysis time in the stressed group significantly prolonged than those in the control group. tissue plasminogen activator activity was also significantly lower in the stressed group. These effects lasted at least for an hour and returned to the control values 24 hours after the stress. Whole blood serotonin levels, which mainly show serotonin contents in platelets, were higher in the stressed group. A negative correlation between whole blood serotonin and tPA activity in the stressed group was obtained. These results suggest that prolonged mental stress impairs fibrinolysis by decreasing tPA activity with a concomitant increase of serotonin contents in platelets.

Medroxyprogesterone acetate (mpa) increases pai-1 secretion from huvec and elevates the plasma-levels of pai-I in-vivo.

Plasma levels of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in patients administered with medroxyprogesterone acetate (MPA) (800 mg/day) or tamoxifen (20 mg/day) were assayed. Active free PAI-1 levels of patients given MPA were significantly higher than those either given tamoxifen or post adjuvant therapy, t-PA antigen levels were not different among the three groups. In in vitro study, treatment of cultured human umbilical vein endothelial cells (HUVEC) with MPA (10(-6)-10(-8) M) decreased PAI-1 levels significantly with a trend to decrease in t-PA levels.

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.

Correlation between serotonergic measures in cerebrospinal fluid and blood of subhuman primate.

The relationship between the concentration of serotonin (5-hydroxytryptamine; 5-HT), its precursor; tryptophan (Trp) and the main metabolite 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid (CSF) and blood of monkey have been studied. 5-HT, Trp and 5-HIAA underwent circadian changes in both CSF and blood. Significant correlations were found between 5-HT, 5-HIAA and Trp in CSF and blood. The significance of these findings and their relationship to the use of peripheral serotonergic system as a functional model of the central nervous system are discussed.

Foot shock-induced changes in blood and brain serotonin and related substances in rats.

The effects of electric foot shock on peripheral and central serotonergic systems in rats have been studied. We have focused on the time course alterations with particular attention being paid to changes in 5-HT, 5-HIAA, tryptophan concentrations and 5-HIAA/5-HT ratios in blood and various parts of the brain, observed within 1 h following stress application. Blood and brain (7 regions) samples were taken immediately after electric foot shock, 30 min, 1 and 24 h later. In the blood stress induced a rise in tryptophan level as well as rises in 5-HT, 5-HIAA levels and 5-HIAA/5-HT ratio within 1 h following stressful treatment. Tryptophan concentration was found to be increased in every part of the brain within 1 h after electric foot shock application. In striatum it remained higher even after 24 h. 5-HT level showed a significant rise only in medulla, while hypothalamus was the sole region where a fall in 5-HT was found. In other parts of the brain 5-HT level remained unaffected by stress. 5-HIAA content increased in almost every brain area studied except cerebellum and striatum. 5-HIAA/5-HT ratios shared the same pattern of changes. Briefly, foot shock altered 5-HT turnover in various brain regions, in particular within the first hour following stress application, whereas delayed response to stress was rarely observed. Increased brain tryptophan level seems to be necessary to cope with the enhanced 5-HT metabolism caused by stress, reflecting as a rise in 5-HIAA concentration and 5-HIAA/5-HT ratio.

Impaired fibrinolysis in hypertension and obesity due to high plasminogen activator inhibitor-1 level in plasma.

In order to elucidate the influence of the risk factors of coronary heart disease on the fibrinolytic activity, relationships between blood pressure, body mass index (BMI), plasma lipoprotein (a) (Lp(a)) level and the plasma levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were analyzed in the subjects with mild hypertension. Systolic blood pressure showed a positive correlation with total PAI-1 and free PAI-1. Diastolic blood pressure showed no correlation with these proteins involved in the fibrinolytic system. BMI had a positive correlation with total PAI-1, free PAI-1 and euglobulin clot lysis time (ECLT). Plasma Lp(a) level showed correlation with neither blood pressure nor fibrinolytic parameters, but it showed weak negative correlation with body mass index (BMI). These results suggest that high blood pressure and obesity tend to increase free PAI-1 which reduces fibrinolytic activity. Lp(a), however, seems not to influence directly the fibrinolytic system but may work to decrease fibrinolytic activity only in conjunction with other risk factors. The effects of daily drinking of alcohol and smoking on the fibrinolytic system were also investigated in the present study and we obtained the results that habitual drinking increased plasma levels of both tPA and PAI-1 whereas smoking did not affect fibrinolytic activity. These results suggest that risk factors for coronary heart disease such as hypertension and obesity are closely related to the impaired fibrinolysis.

Serotonergic measures in blood and brain and their correlations in rats treated with tranylcypromine, a monoamine oxidase inhibitor.

Tranylcypromine, a monoamine oxidase inhibitor, was administered to male Wistar rats in order to investigate its effects on blood and brain serotonin related substances after 1, 4, and 24 h following injection and possible relations between serotonergic measures in central nervous system and periphery. The dose of the drug tested was responsible for an increase in blood serotonin with a simultaneous fall in its metabolite 5-hydroxyindoleacetic acid (5-HIAA) compared to either pretreatment or control values. These changes were the most marked after 4 and 24 h following tranylcypromine injection. Almost all brain areas studied (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus, and cortex) were to be affected by monoamine oxidase inhibitor treatment. They exhibited a rise in serotonin content starting from 1 h after drug administration and lasted in many parts of the brain up to 24 h, which was accompanied by a parallel fall in 5-HIAA level. All these changes were significant when compared to baseline and control values. Alterations in blood serotonin correlated positively with changes in brain serotonin and negatively with brain 5-HIAA, while the opposite pattern of correlations was found regarding blood 5-HIAA and the content of serotonin and 5-HIAA in various brain areas studied. This pattern of correlations speaks in favor of an existence of mutual relations between blood and brain serotonin related substances. Our results suggest that blood serotonin and 5-HIAA may serve as an index of monoamine oxidase inhibitor action on the central serotonergic system.

[Studies on the metabolic fate of isepamicin sulfate (HAPA-B). IV. Intramuscular, intravenous and drip intravenous administration of HAPA-B in dogs].

The plasma concentration and urinary excretion of isepamicin sulfate (HAPA-B) were studied following intramuscular, intravenous and drip intravenous administrations of 6.25, 25 and 100 mg/kg in dogs. Maximum plasma concentrations (Cmax) of HAPA-B after intramuscular, intravenous and drip intravenous administration depended on dosage levels. Biological half-lives (T1/2) and areas under plasma concentration-time curves (AUC) for the three different routes of administration were similar to each other. The peak plasma concentration of HAPA-B achieved with intramuscular administration was similar to that with a 1-hour drip intravenous administration at a dose level of 6.25 or 25 mg/kg. On the other hand, at a dose level of 100 mg/kg, the Cmax following intramuscular administration was similar to that following 2-hour drip intravenous administration. It was, therefore, presumed that the plasma concentration curves which are similar to that of intramuscular administration can be obtained by regulating the infusion time. The observed Cmax value for drip intravenous administration was a little higher than the theoretical Cmax value for drip intravenous administration calculated from parameters for intramuscular administration. Simulation curves obtained for extended infusion times agreed more closely with observed curves than curves simulated for shorter infusion periods. These investigations showed that plasma concentration curves for any dosage levels can be estimated from parameters calculated from experimental data obtained using intramuscular or drip intravenous administration. HAPA-B was rapidly excreted into the urine after administration through any of these 3 routes and 71 approximately 89% of the dose was excreted into the unrine in 24 hours at all dosage levels. Bioautograms of thin-layer chromatographs of the 0 approximately 6 hours urine after intramuscular administration showed single bands with a similar Rf value to that of the standard HAPA-B. No significant differences in plasma concentration and urinary excretion between HAPA-B and amikacin were observed upon intramuscular or intravenous administration of 25 mg/kg.

[Studies on the metabolic fate of isepamicin sulfate (HAPA-B). II. Accumulation of HAPA-B after multiple administration in rats].

The accumulation of isepamicin sulfate (HAPA-B) in tissues and plasma was studied upon multiple intramuscular and intravenous administrations of 25 mg/kg of HAPA-B daily for 8 or 15 days to male rats. Shapes of plasma concentration curves in multiple intramuscular and intravenous administrations were very similar to that in a single administration. Drug concentrations in kidney at 24 hours after 8- and 15-multiple administrations through both routes were 3 to 4 and 4 to 5 times as high as that after a single administration. The concentration in kidney increased by multiple administrations for 8 days, but did not increase so highly there after till the 15-multiple administrations. On the other hand, the elimination of HAPA-B from kidney after multiple administrations was similar to that after a single administration. The accumulation of HAPA-B upon multiple administration was also observed in lung, heart, spleen and liver, but peak concentrations of the drug in these tissues were lower than that in kidney.

[Studies on the metabolic fate of isepamicin sulfate (HAPA-B). I. Absorption, distribution, metabolism and excretion in rats].

Absorption, distribution, metabolism and excretion of isepamicin sulfate (HAPA-B), a new aminoglycoside antibiotic, after a single administration were studied in rats. After intramuscular administration of HAPA-B at a dose level between 6.25 and 100 mg/kg, the drug was rapidly absorbed to reach the peak in 0.10 to 0.21 hour (Tmax). The maximum drug concentration in the plasma (Cmax) and the size of the area under the plasma concentration-time curve (AUC) depended on dose levels. The HAPA-B disappeared rapidly from the plasma after intramuscular and intravenous administrations with biological half-lives (T1/2) from 0.41 to 0.47 hour with intramuscular administration and from 0.23 to 0.35 hour with intravenous administration. Peak time after intramuscular, intraperitoneal and subcutaneous administration of HAPA-B at a dose of 25 mg/kg were 0.18, 0.24 and 0.37 hour, respectively. Maximum drug concentrations in plasma were 64.15, 53.71 and 40.39 micrograms/ml and biological half-lives of the drug were 0.47, 0.73 and 0.87 hour, respectively. The HAPA-B was distributed rapidly into tissues, especially at a high level into kidney after intramuscular or intravenous administration of 25 mg/kg. Concentrations in lung and heart were next to that in kidney, but were not higher than plasma concentrations. The drug was excreted mainly into the urine after intramuscular and intravenous administration within 24 hours and approximately 79 to 90% of the administered amount was excreted. Meanwhile, the excretion of HAPA-B into the bile was 0.1% or less during the first 24 hours after intramuscular and intravenous administration. Bioautograms of thin layer chromatographs of 0 approximately 6 hours urine samples after intramuscular administration showed single bands with the identical Rf value to the standard HAPA-B. No difference between male and female was observed in the fate of the administered HAPA-B through intramuscularly. The shape of the plasma concentration curve and the urinary excretion after intramuscular administration of HAPA-B at the dose of 25 mg/kg was similar to those of amikacin (AMK) and gentamicin. Tissue concentrations after intramuscular and intravenous administration of HAPA-B were also similar to AMK.

[Metabolic fate of 14C-isepamicin sulfate (14C-HAPA-B) in rats. II. Distribution and excretion after the successive administration].

Tissue concentrations of 14C-isepamicin (14C-HAPA-B) and its excretion were studied in male rats after 8- and 15-successive intramuscular administrations of 25 mg/kg/day. Plasma levels were hardly affected by the successive administration, and plasma concentrations as time patterns with 8- and 15-successive administrations were very similar to that with a single administration. Kidney levels of the drug increased remarkably by successive administrations. In all the organs tested except kidney, the elimination of the drug from the tissues tended to become somewhat slower with successive administrations but maximum concentrations were hardly affected by successive administrations. There was little difference between the excretion rates (urinary and fecal) after the successive doses and that after a single dose.

[Metabolic fate of 14C-isepamicin sulfate (14C-HAPA-B) in rats. I. Absorption, distribution, metabolism and excretion after a single intramuscular and intravenous administration].

Absorption, distribution, metabolism and excretion of 14C-isepamicin sulfate (14C-HAPA-B) following a single intramuscular and intravenous administration of 25 mg/kg were studied in male rats. After an intramuscular administration, the drug was rapidly absorbed and the maximum plasma level of about 75 micrograms/ml was obtained at 10 minutes after the administration. The plasma levels rapidly decreased following either intramuscular or intravenous route. The HAPA-B was rapidly distributed into tissues except the central nervous system and the eye ball. Especially high concentrations were attained in kidney and cartilage tissues, concentrations in lung followed these tissues. Radioactivity remained in kidney for a long period, but it disappeared from cartilage and other tissues rapidly. The radioactivity in kidney was located in the cortex at 24 hours after administration. There was no difference in the distribution of radioactivity with different administration routes. The HAPA-B was mainly excreted in the urine following either intramuscular or intravenous administration. Approximately 80% of the dose by intramuscular and 92% by intravenous administrations were excreted during the first 4 hours. Within 24 hours, over 95% was recovered in either routes. The radioautogram of the thin-layer chromatography of the 0 approximately 16-hour urine showed a single radioactive zone with an identical Rf value to HAPA-B. Binding ratios of 14C-HAPA-B to plasma protein were less than 10% both in vitro and in vivo and to erythrocytes less than 9% in vitro.

[Metabolic fate of 14C-isepamicin sulfate (14C-HAPA-B) in rats. III. Placental transfer and excretion into milk].

Placental transfer and excretion into milk of 14C-isepamicin sulfate (14C-HAPA-B) following a single intramuscular or intravenous dose of 25 mg/kg were studied in pregnant or lactating rats. Concentrations of HAPA-B in placenta, ovary and uterus reached their maxima in 10 minutes after administration then declined rapidly. The maximum concentration in the fetal membrane was similar to 10-minute levels in these other tissues, but was attained in 4 hours or later after the drug administration and some drug still remained there even at 24 hours. A small amount of radioactivity was distributed into the fetus and the maximum level in the fetus was attained in 1 approximately 4 hours after administration, much later than in maternal tissues. The concentration in the fetal kidney was the highest in the fetus, but only 1 microgram/g or lower was found. A very small amount of radioactivity was also found in the fetal bone by radioautography. The drug was excreted into milk at 2 approximately 4 micrograms/ml during the first 6 hours and decreased a little in 24 hours after administration. There was no difference in results due to administration routes.