The effect of Biosynthetic Human Proinsulin on glucose uptake by skeletal muscle was studied in the isolated perfused hindquarter of fasted rats. Animals were randomly assigned to the control group, insulin-perfused or proinsulin-perfused group. Glucose disappearance from the perfusate and muscle glycogen levels before and after 2 hours perfusion were measured. Perfusate glucose concentration showed the greatest decline in the insulin group, which was significantly lower (p < .01) than control from 60 to 120 min. Proinsulin perfusion resulted in a smaller and delayed decrease in perfusate glucose. The proinsulin perfusate glucose levels were significantly higher (p < .05) than the insulin glucose values during the second hour of perfusion. After the first hour of perfusion, insulin infusion resulted in higher rates of glucose uptake than control (p < .005) or proinsulin infusion (p < .05). The glucose uptake by muscles perfused with proinsulin was significantly different from control values only at the 2 hour time point (p < .05). Glycogen concentration following insulin infusion increased significantly in the oxidative muscles, i.e. soleus (p < .05) and red vastus (p < .002). These increases in glycogen were significantly different from the changes observed in muscles of control animals. The plantaris and white vastus muscles, which have fast twitch fibers, did not show a significant response to insulin. Proinsulin perfusion decreased glycogen levels regardless of the muscle type. This decline was significantly different from the glycogen changes in soleus (p < .025), plantaris (p < .001) and white vastus (p < .05) muscles of control animals. The proinsulin glycogen fall was also significantly different from the insulin induced response in soleus, plantaris and red vastus muscles (p < .001). These results show that proinsulin has 8.6 % of the biologic potency of insulin for glucose uptake in rat skeletal muscle. Insulin induced an increase in glycogen concentration in oxidative muscles, but proinsulin elicted a drop in glycogen level regardless of the muscle type.

Recent anatomical investigations have revealed that the striatum is an intrinsically heterogeneous structure that forms multiple parallel circuits with different cortical areas. The present series of experiments investigated the possibility that such anatomical diversity may promote functional differences between subregions of the rat dorsal striatum. Using different versions of the water maze and radial arm maze tasks, evidence is presented showing that the medial and lateral subregions of the caudate-putamen make distinct contributions to behaviors guided by either ambient spatial or discrete cues. The findings support a multiple subsystems view of dorsal striatal function. Specifically, the lateral (sensorimotor-innervated) caudate-putamen may mediate the process of habit formation based on simple stimulus-response associations, whereas the medial (limbic-innervated) caudate-putamen may contribute to the cognitive/spatial control of behavior in competitive response situations. Further evidence is presented suggesting that the medial caudate-putamen is functionally related to the hippocampal system. Together, these structures may form a functional limbic circuit that mediates the serial processing of cognitive/spatial information.

The anticonvulsant drug valproic acid (VPA) is extensively metabolized with 16 metabolites identified in man. Of interest are the unsaturated metabolites which appear to be responsible for the observed secondary antiepileptic activity and/or idiosyncratic hepatotoxicity. A study by Abbott et al . (1986) has shown 2-((E)-1'-propenyl)-(E)-2-pentenoic acid ((E,E)-2,3'-diene VPA) to be a major unsaturated metabolite of VPA. Acheampong (1985) demonstrated that an isomeric mixture of 2,3'-diene VPA prevented pentylenetetrazole-induced seizures in mice and had 60% the potency of VPA. Research on (E,E)-2,3'-diene VPA is limited even though the diene appears to be a contributor to the secondary antiepileptic effect. Available synthesis of 2,3'-diene VPA result in two or more isomers with very low yields as shown by Acheampong and Abbott (1985). The object of this work was, therefore, to synthesize (E,E)-2,3'-diene VPA in sufficient quantity and isomeric purity for metabolic and pharmacokinetic studies in rats. Synthesis of (E,E)-2,3'-diene VPA was achieved by the alkylation of ethyl (Z)-2-pentenoate to afford ethyl 2-(l'-hydroxypropyl)-(E)-2-pentenoate. Dehydration using methanesulfonyl chloride and potassium hydride gave the ethyl ester of (E,E)-2,3'-diene VPA. Hydrolysis of the ester in 1 N NaOH afforded an 81.2% pure sample of (E,E)- 2,3'-diene VPA as determined by GCMS and NMR. A second method was used to unequivocally synthesize (E,E)-2,3' - diene VPA whereby an O-trimethylsilylketene acetal was oxidized via a hydride abstractor to yield two isomers of 2,3'-diene VPA plus the starting material (E)-3-ene VPA. The identity of the products were determined by GCMS with the major isomer being (E,E)-2,3'-diene VPA. In the metabolism studies, Wistar rats were given 100 mg/kg i.p. of (E,E)-2,3'-diene VPA and bile and urine collected for 24 hours. GCMS analysis revealed three metabolites present in both bile and urine. These were reduction products of (E,E)-2,3'-diene VPA metabolism and consisted of the monounsatured (E)-3-ene VPA and (E)-2-ene VPA plus the fully saturated VPA. These results suggest that trace levels of (E)-3-ene VPA observed after VPA dosing may not be a direct metabolic product of VPA itself but rather a reduced metabolite of (E,E)-2,3'-diene VPA. All polar metabolites are yet to be identified. For the pharmacokinetic studies, two doses, 20 and 100 mg/kg of (E,E)-2,3'-diene VPA were administered i.v. to Wistar rats and the plasma concentration vs time decline curve determined using GCMS analysis of the plasma samples. The bile duct of these animals was then cannulated and the study repeated. A comparison between the elimination rate constant (KE), clearance (C1), and volume of distribution (Vd) between the bile duct intact and cannulated rats for both dose groups revealed no significant differences (p>0.1). A comparison of the KE, Vd, and C1 between dosage groups of both bile duct intact and cannulated rats revealed no significant difference (p>0.08-0.7). Therefore, extensive enterohepatic recirculation was not apparent and the elimination of (E,E)-2,3'-diene VPA appeared to be dose-independent. The diene seems to follow a simple one-compartment model with a half-life of 35.9±8.9 (S.D.) minutes and a large volume of distribution of 0.95±0.22 (S.D.) L/kg. In vitro protein binding studies revealed that (E,E)-2,3'-diene VPA saturates plasma proteins between concentrations of 30 - 600 ug/mL. The percent of (E,E)-2,3'-diene VPA bound decreases from 92.1% to 28.7% as the concentration of diene increases suggesting concentration-dependent protein binding. The synthesis of (E,E)-2,3'-diene VPA in substantial quantities has allowed metabolic and pharmacokinetic studies to be performed in rats. Preliminary studies showed that (E,E)-2,3' -diene VPA was metabolically reduced to monounsaturated and saturated products. Pharmacokinetic data appear to indicate a potential for the diene to accumulate in the central nervous system. Further studies are required to determine the contribution of (E,E)-2,3'-diene VPA to the secondary antiepileptic activity of VPA. / Pharmaceutical Sciences, Faculty of / Graduate

Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

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published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy