Pretreatment with a deleted form of hepatocyte growth factor (dHGF) prevents the mortality of plasma-loss-induced hypovolemic shock in rats.

Severe trauma, infection, burn, pancreatitis and major surgery often induce circulatory collapse leading to multiple organ failure and death. It is hypothesized that therapy for the attenuation of circulatory collapse may improve the prognosis in these diseases. Previous work has documented that pretreatment with a deleted form of hepatocyte growth factor (dHGF) in normal rats increases the circulating plasma volume that reflects its accelerating action of hepatic protein synthesis. Therefore, the effects of pretreatment with dHGF on hypovolemic shock models were studied in rats. Rats were intravenously administered dHGF (1 mg/kg, twice daily for 5-6 days) or vehicle, and subjected to a 25% total body surface area full-thickness burn or a trypsin-induced acute pancreatitis. In rats that were receiving vehicle, survival rates on day 7 after injury induction were 12% in the burn model and 5% in the pancreatitis model, respectively. In both models, hematocrit values were apparently increased and circulating plasma volumes were decreased compared to sham-operated rats at 6 h after injury induction. The pretreatment of animals with dHGF increased the survival rates on day 7 to 40% in the burn model and 29% in the pancreatitis model. dHGF-treatment in normal rats decreased the hematocrit values and increased the circulating plasma volumes, and these changes of hematocrit value and circulating plasma volume were also maintained after injury induction. These findings suggest that dHGF pretreatment prevents the mortality in the severe burn and acute pancreatitis, and that its effect may contribute to ameliorating the progressing of plasma-loss-induced hypovolemia.

(+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate (SNI-2011, cevimeline hydrochloride) induces saliva and tear secretions in rats and mice: the role of muscarinic acetylcholine receptors.

We investigated effects of (+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] hydrochloride, hemihydrate (SNI-2011, cevimeline hydrochloride), a rigid analogue of acetylcholine, on saliva and tear secretions in rats and mice to evaluate its therapeutical efficacy for xerostomia and xerophthalmia in patients with Sjogren's syndrome and X-ray exposure in the head and neck. Intraduodenal administrations of SNI-2011 increased saliva secretion in a dose-dependent manner at doses ranging from 3 to 30 mg/kg in normal rats and mice, two strains of autoimmune disease mice and X-irradiated saliva secretion defective rats. The salivation elicited by SNI-2011 was completely inhibited by atropine. A similar atropine-sensitive response was observed in tear secretion. In rat submandibular/sublingual gland membranes, [3H]quinuclidinyl benzilate (QNB) binding was saturable, and Scatchard plot analysis revealed a single population of binding sites with a Kd of 22 pM and a maximal binding capacity of 60 fmol/mg protein. The competitive inhibition curve of the [3H]QNB binding by SNI-2011 was obtained, and its dissociation constant value calculated from IC50 was 1-2 microM. These results suggest that SNI-2011 increases saliva and tear secretions through a direct stimulation to muscarinic receptors in salivary and lacrimal glands, and they suggest that SNI-2011 should be beneficial to patients with Sjögren's syndrome and X-ray exposure in the head and neck.

Modulation of rhythmical slow activity, long-term potentiation and memory by muscarinic receptor agonists.

We investigated the cholinergic modulation of hippocampal rhythmical slow activity (or theta activity), long-term potentiation and a behavioral memory task. The intravenous administration of the muscarinic receptor agonists, AF102B ((+/-)-cis-2-methyl-spiro(1,3-oxathiolane-5,3') quinuclidine hydrochloride hemihidrate) and oxotremorine, induced rhythmical slow activity at doses of 1.0 mg/kg and 0.01 mg/kg, respectively. Long-term potentiation of population spike amplitude in the hippocampal CA1, which was induced by tetanic stimulation to the Schaffer collateral/commissural fiber, was increased by AF102B (1.0 mg/kg i.v.) and oxotremorine (0.01 mg/kg i.v.). Oral administration of AF102B and oxotremorine improved scopolamine-induced memory deficits in a passive avoidance task in mice at doses of 1.0 mg/kg and 0.2 mg/kg, respectively. The correspondence of the effective doses of muscarinic receptor agonists in these three experiments suggested the cholinergic correlation of rhythmical slow activity, long-term potentiation and memory.

Inhibitory action of N omega-nitro-L-arginine methyl ester on in vivo long-term potentiation in the rat dentate gyrus.

Recent in vitro observations have led to the suggestion that nitric oxide (NO) plays a modulatory role in the expression of long-term potentiation (LTP). We investigated whether an NO synthesis inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), affects the generation of LTP in anesthetized rats in vivo. Administration of L-NAME (0.1 and 1 nmol, i.c.v.) suppressed the magnitude of LTP dose dependently in the dentate gyrus of anesthetized rats. Co-injection of L-arginine, which interferes with the inhibitory action of L-NAME on NO synthesis, reversed these effects, whereas there was no reversal in rats that received co-administration with D-arginine. These results provide further support for the hypothesis that NO plays a modulatory role in the expression of synaptic potentiation.

Prostaglandin E1 enhances hepatic portal venous flow by dilating the portal vascular bed in 70% hepatectomized dog.

The effects of portal, hepatic arterial and femoral venous administration of prostaglandin E1 (PGE) on portal venous flow (PVF) and hepatic arterial flow HAF were examined before and after 70% hepatectomy in anesthetized dogs. In the hepatectomized condition, portal venous administration of PGE (0.5 microgram/kg/min) caused an increase in PVF without any change in systemic arterial pressure (SAP). HAF was unchanged following the injection. The portal effect of PGE on PVF was dose-dependent, and a reduction in portal venous resistance was seen. However, the same dose of PGE failed to change PVF under intact liver conditions. Hepatic arterial administration of PGE (0.5 microgram/kg/min) brought no significant change in PVF or HAF, with or without hepatectomy. Femoral venous administration of PGE (0.5 micrograms/kg/min) produced an increase in PVF concomitant with a significant decrease in SAP. HAF showed no change after the injection. A decrease in PVR was seen only in the hepatectomized condition. It is concluded that PGE is potent in increasing PVF in the hepatectomized condition, and the portal vasculature is involved as the site of action.

Effects of a M1 muscarinic receptor agonist on the central cholinergic system, evaluated by brain microdialysis.

The effects of a novel M1-receptor agonist, AF102B (FKS-508; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine), on the central cholinergic system in vivo were evaluated by determination of acetylcholine (ACh) content in the rat brain after microwave irradiation and by measurement of ACh release with microdialysis perfusion in freely moving rats. Intraperitoneal administration of AF102B resulted in a significant decrease of ACh content in the brain, while AF102B produced an increase of in vivo ACh release. The present results suggest that ACh content in the brain after treatment with muscarinic agents may be related to the changes of ACh release, in which both M1 and M2 muscarinic receptors may be involved.

Beneficial effects of FKS-508 (AF102B), a selective M1 agonist, on the impaired working memory in AF64A-treated rats.

The effects of FKS-508 [AF102B; cis-2-methylspiro(1,3-oxathiolane-5,3')quinuclidine], a selective M1 muscarinic receptor agonist, were examined to predict the possible activity on memory disorders using a T-maze and radial-arm maze task in experimental amnesia models. The amnesia models were produced by bilateral intracerebroventricular injection of ethylcholine aziridinium ion (AF64A), a selective cholinotoxin, in rats. Repeated administrations of FKS-508 (5 mg/kg/day, i.p.) for 5 weeks significantly ameliorated impaired performance of AF64A-treated rats (AF64A-rats) in a delayed alternation task in the T-maze. Repeated administrations of FKS-508 (1 and 5 mg/kg/day, p.o.) for 5 weeks significantly ameliorated acquisition failures of AF64A-rats in a radial-arm maze task. Single administration of FKS-508 (1 and 5 mg/kg, p.o.) significantly reduced the incorrect choices of AF64A-rats in a radial-arm maze task with 6 hr-delay time. No abnormalities in general behaviors, such as loss of appetite and ataxia, were observed in rats treated with FKS-508 repeatedly during 5 weeks. Our present results showed that FKS-508 can ameliorate memory impairments in AF64A-rats with central cholinergic hypofunction without causing any behavioral abnormalities. FKS-508 may be considered as a candidate for the clinical examination of the cholinergic hypothesis of senile dementia of the Alzheimer type.

Antiulcerogenic compounds isolated from Chinese cinnamon.

Two active compounds that prevent serotonin-induced ulcerogenesis in rats were isolated from Chinese cinnamon (the stem bark of Cinnamomum cassia) and identified as 3-(2-hydroxyphenyl)-propanoic acid and its O-glucoside. The former compound, administered orally or parenterally to rats at a remarkably low dose (40 micrograms/kg body weight), also inhibited gastric ulcers induced by the other ulcerogens such as phenylbutazone, ethanol, and water immersion stress, although it failed to prevent indomethacin-induced ulcers. Pharmacological studies have shown that 3-(2-hydroxyphenyl)-propanoic acid hardly inhibited the secretion of gastric acid, but promoted the gastric blood flow. These results suggest that the antiulcerogenic effect of this compound is probably attributable to the potentiation of defensive factors through the improvement of the circulatory disorder and gastric cytoprotection.

Amelioration of experimental amnesia (passive avoidance failure) in rodents by the selective M1 agonist AF102B.

Effect of AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine) on experimental amnesia was examined using a passive avoidance task in rodents. The amnesia was produced by anti-cholinergic agents, AF64A (intracerebroventricularly) and scopolamine (subcutaneously). AF102B ameliorated the memory deficits in AF64A-treated rats at 0.1-1 mg/kg, i.p. and at 1-5 mg/kg p.o. and in scopolamine-treated mice at 1-10 mg/kg, i.p. These results suggest that AF102B may compensate for central cholinergic defects and could be developed as a possible therapeutic drug for senile dementia of the Alzheimer type.

Effects of intracerebroventricular injection of AF64A on learning behaviors in rats.

The effects of intracerebroventricular (ICV) injection of ethylcholine aziridinium ion (AF64A) (3 nmole/2 microliter, each lateral ventricule), a putative selective cholinotoxin, on learning behaviors and choline acetyltransferase (ChAT) activity were studied in rats. AF64A-treated rats (AF64A-rat) exhibited deficient performance in a passive avoidance task and a delayed alternation task in the T-maze, but demonstrated superior avoidance response in a two-way shuttle avoidance task. These changes in learning behaviors were associated with the selective decrease of hippocampal ChAT activity. Physostigmine (0.1 mg/kg, i.p.) significantly improved the retention latency of AF64A-rats in the passive avoidance task. AF64A-rats receiving physostigmine (0.2 mg/kg, i.p.) exhibited a slight but not significant improvement of performance in the delayed alternation task in the T-maze. These findings suggested that ICV injection of AF64A may be useful for producing an experimental amnesia model with hippocampal cholinergic hypofunction like Senile dementia of the Alzheimer type (SDAT), if appropriate learning tests are selected.

Thrombolytic effect of single-chain pro-urokinase in a rabbit jugular vein thrombosis model.

The thrombolytic effect of single-chain pro-urokinase (SCPU) was examined in the rabbit using a jugular vein thrombosis model. Infusion of a low dose (120,000 IU/kg) of either urokinase (UK) or SCPU did not produce any significant thrombolysis. However, UK administration at such a low dose caused 20% degradation of circulating fibrinogen. A high dose (480,000 IU/kg) caused significant thrombolysis. The degree of fibrinogenolysis was about 20% in SCPU, but about 80% in UK. The thrombolytic efficiency of SCPU was thus about 3 times larger than that of UK. Analysis of fibrinolytic parameters such as plasminogen, alpha 2-plasmin inhibitor, etc. suggested that UK caused systemic activation of the fibrinolytic system, but SCPU, locally limited activation on the fibrin surface (fibrinolysis). These results indicate that SCPU represents a highly efficient thrombolytic agent without producing fibrinogenolysis.

Human urinary kallikrein (HUK): large-scale purification and direct solid-phase radioimmunoassay (RIA).

HUK was purified from 1,000 liters of fresh urine by the following procedures: silica gel adsorption, gel filtration on Sephadex G-75, DEAE-Sephadex chromatography, bentonite treatment, affinity chromatography on aprotinin-Sepharose 4B, and rapid gel filtration on a TSK Gel G-3000 SWG column. Seventeen mg of HUK being found to be pure by means of various analyses was obtained. The pI values of the heterogeneous components of HUK were 3.5, 3.8, and 4.1, while the corresponding molecular weights of these components were 5.4 X 10(4), 4.9 X 10(4), and 4.4 X 10(4), respectively. The antigens (125I- and non-labeled HUK) were incubated for 4 hrs at 37 degrees C in polystyrene test tubes to which anti-HUK rabbit IgG had been immobilized. The quantitative range of the standard curve was 1-128 ng. This Ria principally recognized active form of HUK. Therefore, total and inactive HUK also could be determined by the combination of the RIA with trypsin treatment of the urine sample. The RIA correlated closely with both S-2266 amidolytic assay and kininogenase assay.

Kinetics of urokinase-induced thrombolysis in a biphasic in vitro system.

The role and effect of added lys-plasminogen (lys-PLG) on urokinase-induced thrombolysis in an in vitro biphasic system were investigated. The kinetics of lysis of whole blood thrombi was followed in perfusion mediums of normal plasma, PLG-deficient plasma and normal saline using a high and a low concentration of urokinase (UK). The lysis of standard whole blood thrombi in whole blood perfusion mediums to which had been added UK alone or UK plus lys-PLG was compared to whole blood thrombi enriched with lys-PLG by incorporation during thrombus formation or by adsorption during perfusion. In addition, the kinetics of lysis of PLG-deficient fibrin thrombi perfused in PLG-deficient plasma or normal saline was studied when lys-PLG had been added to the thrombus, to the perfusion medium or to both thrombus and medium. In PLG-deficient plasma from which plasmin inhibitors had not been removed, thrombolysis was minimal even at a high concentration of UK. This effect could be neutralized, and to some extent, regulated, by lys-PLG enrichment of the medium. Both PLG-incorporated and PLG-adsorbed whole blood thrombi gave initial and sustained acceleration of UK-induced lysis in comparison with standard nontreated thrombi. It is concluded that in a blood-thrombus biphasic thrombolytic system induced by UK, there is interaction between the phases, and that PLG in both phases influences thrombolysis.

Molecular exchange between blood and in vitro thrombi.

Uptake of radioiodinated Lys-plasminogen (125I-PLG), albumin (125I-ALB), and tritiated water (3H-H2O) by in vitro thrombi and interchange of these molecules with blood in an in vitro perfusion system were investigated. The radioisotopes were taken up by thrombi either by incorporation during formation in radiolabelled blood or by perfusion of preformed nonradioactive thrombi in radiolabelled blood. Release of the radioisotopes into a perfusion medium of nonradiolabelled blood was then monitored over a 120-min period. The small molecules of 3H-H2O were rapidly released from the thrombi and achieved equilibrium with the perfusion medium by 120 min in that the specific radioactivity (cpm/mg) of the thrombi equalled that of the medium. The larger molecules of 125I-PLG and 125I-ALB were more slowly released and did not reach equilibrium with the perfusion medium over the period studied. Release of 125I-ALB was intermediate between that of 3H-H2O and 125I-PLG. The greater retention of 125I-PLG by the thrombi was consistent with the high binding affinity of plasminogen for fibrin. The demonstrated movement of these radioisotopes between medium and thrombus suggests that thrombi exist in blood in a dynamic state of flux, exhibiting a fluid exchange of molecules between the interstitial compartment of the thrombus and blood.

Uptake of 125I-Lys-plasminogen by in vitro thrombi.

The specificity, distribution and rate of uptake of radiolabelled 125I-Lys-plasminogen by in vitro thrombi was investigated. 125I-Lys-plasminogen was added to whole blood perfusion mediums containing preformed thrombi and to whole blood prior to thrombus formation. Uptake was assessed by means of radioisotopic analysis and autoradiography. The plasminogen was taken up by thrombi during and after their formation. The largest percentage was in the fibrin component. epsilon-Aminocaproic acid-blocking experiments confirmed the specificity of plasminogen binding to fibrin. Autoradiography of the thrombi revealed plasminogen in the RBC-fibrin part and in platelet-fibrin aggregates. Plasminogen uptake and penetration into preformed thrombi were found to increase as a function of time. However, formation of thrombi from plasminogen-enriched blood was a more effective means for increasing the plasminogen content of thrombi than perfusion of preformed thrombi in a plasminogen-enriched medium, over the time period studied.

Molecular structure of taka-amylase A. I. Backbone chain folding at 3 A resolution.

The crystal structure of Taka-amylase A was studied by an X-ray diffraction method at 3 A resolution. A total of 452 amino acid residues were found from the electron density map at the present stage. The four disulfide bonds and the branched carbohydrate were also located on the map. The difference electron density map of the maltotriose-soaked crystal showed that a maltose unit was bound in the active center left. The binding of iodine atoms to the enzyme was also studied.