Consequences of injury to peripheral nerves and opioid tolerance share features including tactile and thermal hypersensitivity, decreased spinal opioid antinociception and upregulation of spinal dynorphin. Dynorphin may normally produce antinociception (opioid effect) but may be pronociceptive in pathological states (through direct or indirect actions at NMDA receptors). Sustained morphine, but not placebo, exposure elicited a time-related onset of both tactile and thermal hypersensitivity along with antinociceptive tolerance to i.th. morphine. Spinal dynorphin levels were significantly increased following morphine exposure. Treatment with either i.th. MK-801 or antiserum to dynorphin (Dyn A/S) reversed both morphine-induced abnormal pain and antinociceptive tolerance to spinal morphine. Control pre-immune control serum had no effect. Sustained administration of either DAMGO or (-)-oxymorphone (active form), but not (+)-oxymorphone (inactive form) or vehicle, produced both tactile and thermal hypersensitivity along with antinociceptive tolerance to i.th. agonist challenge. Spinal dynorphin levels were significantly increased following treatment with either DAMGO or (-)-oxymorphone. The possibility that opioid-induced central changes might mediate increased excitability to the spinal cord was tested. Tactile and thermal hypersensitivity was observed at 7, but not 1, day after subcutaneous morphine pellet implantation; placebo pellets produced no effects. Basal and capsaicin-evoked release of CGRP was measured in minced spinal cord tissues taken from naive rats or from rats on post-pellet days 1 and 7. The content and evoked release of CGRP was significantly increased in tissues from morphine-exposed rats at 7, but not 1, day after implantation. Prodynorphin "knock-out" (KO) and wild-type (WT) mice were studied for changes in sensitivity to non-noxious mechanical and noxious radiant heat after morphine or placebo pellet. After 5 days of treatment, WT, but not KO, mice developed antinociceptive tolerance. Morphine, but not placebo pellet produced a time-related increased sensitivity to non-noxious and noxious stimuli in WT, but not in KO, mice. These data suggest that sustained morphine induces plasticity in both primary afferents and the spinal cord, including increased CGRP and dynorphin content. Morphine-induced elevation of spinal dynorphin content depends on descending influences and enhances stimulated CGRP release. (Abstract shortened by UMI.)

Prostaglandin F2 alpha (PGF₂α) regulates physiological responses including lowering of intraocular pressure, luteolysis, and parturition. FP prostanoid receptors are GPCRs mediating the actions of PGF₂α. mRNA splice variants exist for the ovine FP receptor gene and are designated FP(A) and FP(B). Thus far, receptor heterogeneity for the human FP receptor gene has yet to be established. This dissertation identifies human FP receptor isoforms and their pharmacological significance. Utilizing PCR, a putative human FP(B) (hFP(B)) ortholog from placenta and CX-1 (colon adenocarcinoma) cDNA was identified. These clones produced similar cDNA sequence both containing inverted repeat sequences. However, the mechanism by which the hFP(B) is produced appears different from alternative mRNA splicing and remains unexplained. Pharmacological characterization of the hFP(B) ortholog is ongoing and its significance remains unknown. Additionally, we report the cloning of a FP receptor mRNA splice variant from human heart and placenta cDNA, named human FP sevenless (hFP(S)). The cDNA encoding hFP(S) has a 71 base pair insert that produces a frame shift resulting in a truncated receptor lacking transmembrane-7 and a carboxyl tail. This sequence is identified as a distinct exon localized on the human FP receptor gene. hFP(S) mRNA are expressed in human skeletal muscle, heart and placenta. Immunohistochemical staining showed positive immunoreactivity in vascular endothelium, trophoblast, and decidual cells from placenta. hFP(S) represents the first confirmed alternative splice variant of the human FP prostanoid receptor gene, however, its function is unknown. Pharmacological characterization of hFP(S) demonstrated no significant PGF₂α binding or PGF₂α-mediated inositol phosphate hydrolysis. FLIPR high throughput screening membrane potential assays yielded four potential agonists for hFPS activation that were not observed in (-)293-EBNA cells; oxytocin, PGB₂, AGNA9B9, and AGNA10B10. These potential agonists require further investigation for hFP(S) selectivity and until such data is determined, hFP(S) should continue to be considered an orphan receptor. In conclusion, these studies demonstrate one FP receptor splice variant of the six transmembrane (6TM) nature exists in humans. In addition, preliminary evidence suggests the existence of a hFP(B) receptor ortholog potentially generated from a mechanism other than traditional mRNA splicing and could contribute to the development of colon carcinogenesis.

The passage of peptides across the blood-brain or blood-cerebrospinal fluid barrier is extremely limited. Peptides can be hindered from entering the central nervous system due to the hydrophilic nature of peptides and their susceptibility to enzymatic degradation by various peptidases. This limitation can be overcome through chemical modifications of opioid peptides with the goal of increasing biological stability and blood-central nervous system permeation. In the present studies, an in vitro bovine brain microvessel endothelial cell model of the blood-brain barrier was characterized both functionally and enzymatically. This primary culture model was found to be reflective of the in vivo blood-brain barrier in reference to predicting a peptides relative lipophilicity. Bovine brain microvessel endothelial cells were also found to be quite active enzymatically as far as the peptidases known to be involved in the degradation of methionine enkephalin. The conformationally stable analog of methionine enkephalin, DPDPE, was also characterized for its ability to enter the CNS using the in situ brain perfusion technique. DPDPE was found to enter the brain by both saturable and non-saturable uptake mechanisms. Chlorohalogenation was also found to significantly improve the central nervous system entry as well as biological stability of a potent opioid agonist, biphalin. In addition, the mu-opioid receptor selective antagonist, CTAP, was also evaluated for its ability to enter the CNS. The amount of CTAP that crossed both the blood-brain and blood-cerebrospinal fluid barrier was quantitatively comparable to the mu-selective agonist, morphine. Biphalin was found to enter both spinal and supra-spinal sites that have been shown previously to express mu and delta opioid receptors. In situ brain perfusion experiments identified a saturable component that contributes to the brain entry of [¹²⁵125I-Tyr¹]biphalin. Further experiments revealed that [¹²⁵125I-Tyr¹]biphalin was entering the CNS by the large neutral amino acid transporter and not by the leucine enkephalin uptake system or DPDPE transport system. This research has provided important preliminary work for the characterization of peptide transport into the brain. The importance of using neuropharmaceutical drug delivery vectors in modern medicine needs attention for the evolution of successful drug design targeted for CNS entry.

This work describes (1) the identification of alkyl and aryl 2-imidazolyl disulfides that inhibit growth in human cancer cell lines, (2) examines the antitumor activities of these disulfides in xenografted scid mice, (3) characterizes the chemopreventive activity in min mice of the disulfides IV-2, and (4) suggests the potential mechanisms by which these compounds mediate their actions. The alkyl and aryl 2-imidazolyl disulfides are inhibitors of the TR/Trx which regulates cell growth in both normal and cancer cells and is constitutively activated in a number of human primary cancers (Berggren, 1996). We found that all of the disulfides tested were potent inhibitors of growth in a panel of 60 cancer cell lines tested by the NCI. Disulfides IV-2, VI-2, and DLK-36 were found to inhibit growth of HL-60 leukemia and MCF-breast cancer cells implanted in scid mice. However, these disulfides had no significant inhibitory effect on the growth of human HT-29 colon cancer cells implanted in scid mice. Disulfide IV-2 also demonstrated a chemopreventive activity evaluated in min mice fed diet supplemented with 250 ppm IV-2. These disulfides may function through modulation of cellular redox status. It was determined that disulfides IV-2 and DLK-36 decreased GSH levels and that this effect may be due to indirect inhibition of the TR/Trx redox system or a direct reaction of GSH with the disulfides. Importantly, Human Trx regulates transcription factors such as NF-KB and activation of this transcription factor protects cells from apoptosis. Disulfide IV-2 was observed to inhibit the transcriptional activity of NF-KB. This inhibitory activity may sensitize cells to apoptosis. Most of the disulfides tested induced apoptosis at concentrations of twice the IC50 in both MCF-7 breast and HT-20 colon cancer cells. Interestingly, a protein which is secreted by MCF-7 cells into the media can degrade disulfide IV-2. This putative molecule has a molecular weight (m.w) greater than 3000 and is likely redox sensitive because ultra-filtered conditioned media (3000 m.w.) or aged media drastically decreased the degradation or sequestration activity of conditioned media. Plasma from mice treated with disulfides IV-2 or DLK-36 (i.v.) gave the same HPLC profile as seen in in vitro studies. These disulfides could be inhibiting tumor growth by inhibition of the TR/Trx system and/or depletion of GSH. Finally, the p53 tumor suppressor protein appears to be regulated by redox conditions. Recent, evidence suggests p53 may be regulated specifically by the TR redox system. The rat embryo fibroblast cell line, Al-5, expressing wild-type p53 was less sensitive to disulfides than these same cells expressing mutant p53.

The objective of these studies was to isolate and characterize novel members of the 3A subfamily of canine cytochromes P450. This subfamily is of interest due to the wide range of endogenous and exogenous compounds metabolized. Previous studies led to the identification, isolation, and heterologous expression of 3A12, the major P450 form in canine liver. This enzyme demonstrated the ability to metabolize steroidal compounds and macrolide antibiotics in reconstituted systems. However, several lines of evidence suggested the presence of additional 3A enzymes with different substrate specificities. Initial experiments employed the same library used to clone the 3A12 cDNA and led to the isolation of a cDNA encoding P450 3A26. This new cDNA encoded a protein of 503 amino acids with 33 nucleotide differences encoding 22 amino acid variations compared with 3A12. Immunoblots indicated that 3A26 comigrates with a previously unidentified 3A protein in PB-induced canine microsomes. The 3A26 cDNA was modified for heterologous expression and cloned into the pSE380 expression vector. Expression of 3A26 and 3A12 in E. coli was accomplished using slightly modified protocols developed in this laboratory. Steroid hydroxylase assays indicated that these two enzymes have distinct catalytic profiles, with 3A12 exhibiting a rate of 6β-hydroxysteroid product formation ranging from 4 to 50-fold higher than 3A26. The vast differences in the activities of 3A12 and 3A26 in contrast to their similarity in structure made these enzymes an excellent model system for the identification of structure-function relationships. Studies were done to identify which of the 22 amino acid residue differences between 3A12 and 3A26 confer differences in rates of hydroxylation of progesterone, testosterone and androstenedione. Ten different 3A12/3A26 chimeras were generated using restriction endonuclease sites. Hydroxylase assays indicated that the first four residue changes and the six differences found within an internal PstI fragment were at least partially responsible for differences in the hydroxylation rates of all three steroids tested. Site-directed mutagenesis revealed the importance of 3A26 residues Ile-187, Ser-368, and Val-369. Conversion of these positions to 3A12 residues increased the rates of 6β-hydroxysteroid formation by 10-20 fold for progesterone, testosterone, and androstenedione. These studies identified a new member of the P450 3A subfamily and are the first to use catalytically distinct cytochromes P450 3A from the same species in the elucidation of 3A structure-function relationships.

Prostaglandin F₂(α) (PGF₂(α)) is a locally acting hormone derived from arachidonic acid that is involved in a diverse range of physiological functions including regulation of the corpus luteum and regulation of intraocular pressure. The goal of this research has been to characterize the signaling pathways activated by PGF₂(α). A receptor for PGF₂(α), the FP receptor, had been cloned from a number of species. Based on the cloning of other prostanoid receptors, we hypothesized that isoforms, or alternative splice variants of the FP receptor that differed in their functional coupling might exist. Using a corpus luteum library, we cloned a novel, truncated form of the FP prostanoid receptor, known as the FP(B) receptor isoform. To examine differences in signaling between the FP receptor isoforms, we have generated stable cell lines that express the original FP receptor, known as the FP(A) isoform and cells that express the FP(B) isoform. To date, no differences in the functional coupling between the receptor isoforms have been found. However, during the second messenger studies, we discovered that activation of either receptor isoform leads to changes in the cell morphology and in the cell cytoskeleton. Thus, treatment of the stable cell lines with PGF₂(α) leads to the retraction of cellular projections, and the boundaries between the cells appear less distinct. There is concomitant formation of actin stress fibers and increased tyrosine phosphorylation of p125 FAK. Understanding the molecular mechanisms underlying the PGF₂(α) induced changes in the cell morphology and in the cell cytoskeleton may be relevant for developing better treatments for glaucoma and other diseases.

Since the cloning of the only known cannabinoid receptor in the human brain, CB₁, scientists have been trying to elucidate the mechanisms of action of this receptor. The possibility of yet unidentified cannabinoid receptors in human brain makes it important to be certain that any results obtained from studies of the CB₁ receptor are due strictly to this receptor. Therefore, the cloned human cannabinoid CB₁ receptor has been stably expressed in Chinese Hamster Ovary (CHO/CB₁) cells, which do not endogenously express and known cannabinoid receptors. Saturation binding studies using [³H]SR141716A and [³H]CP55,940 determined that this system expresses the human cannabinoid CB₁ receptor at approximately 1.8 pmoles of receptor/mg protein and that this expression system is able to differentially bind agonists (CP55,940) and antagonists (SR141716A). In the [³⁵S]GTPγS binding assay, CP55,940, WIN 55,212-2 and HU-210 are full agonists, methanandamide and 11-OH-Δ⁹-THC are partial agonists, Δ⁹-THC is a competitive antagonist and SR141716A and AM630 are inverse agonists. The results for the full and partial agonists are consistent with those reported for these: compounds in mammalian brain. The findings that Δ⁹-THC is a competitive antagonist and that SR141716A and AM630 are inverse agonists in the [³⁵S]GTPγS assay are novel. CP55,940 and WIN 55,212-2 were also shown to be full agonists by maximally inhibiting forskolin-stimulated cAMP formation in CHO/CB₁ cells. The finding that SR141716A is an inverse agonist was further supported by its ability to augment forskolin-stimulated cAMP formation. Interestingly, Δ⁹-THC was a partial agonist in this assay since it inhibited forskolin-stimulated cAMP formation, but to a lesser extent than either CP55,950, or WIN 55,212-2. This difference in the activity of Δ⁹-THC warrants further study. Pertussis toxin abolished the effects of cannabinoid ligands in these functional assays, confirming that the effects of these compounds are mediated through inhibitory Gᵢ/ₒ proteins. The data obtained from radioligand binding and functional assays with the human cannabinoid CB₁ receptor stably expressed in Chinese hamster ovary cells conclude that this is a model system to study the pharmacological effects of cannabinoids and to predict the actions of cannabinoids in man.

Uptake and distribution of peptides into the central nervous system (CNS) is limited by a number of factors, central to which is the blood-brain barrier (BBB). Peripheral influences: blood-flow, protein binding in blood, clearance, and metabolism, also greatly affect the ability of peptide drugs to enter the CNS. Nevertheless, the BBB is frequently the rate-limiting factor in peptide drug permeation into the brain. The use of chemical modification, or pharmacological manipulation of the BBB, can improve CNS uptake of peptide drugs. In this examination, methods of peptide distribution, lipophilicity, protein binding, stability, receptor binding, clearance, and BBB permeation were used to assess the affects of various strategies on the delivery a model opioid peptide, DPDPE. DPDPE is a well characterized delta-opioid peptide analogue of met-enkephalin. Characterization of DPDPE uptake at the BBB was assessed both in vitro, using primary culture bovine brain microvessel endothelial cells, and in situ, using brain perfusion analysis in the rat. The first aspect of this examination was aimed at assessing lipophilicity and stereoselectivity via tri-methylating DPDPE, thereby enhancing lipophilicity and creating four distinctive diastereoisomer configurations. Each diastereoisomer was assessed for protein interaction, lipophilicity, BBB permeation ( in vitro & in situ), receptor-binding affinity, metabolic stability and end analgesic effect. Significant variation was shown between the parent form and methylated diastereoisomers, as well as significant variation between each respective diastereoisomer in relation to analgesia and BBB penetration. The second aspect of the examination was aimed at PEGylating [conjugation of a poly(ethylene glycol) to a drug] DPDPE, with focus on BBB permeability characterization. Pharmacokinetic and pharmacodynamic properties were also assessed. PEGylation resulted in significantly decreased clearance, with increased drug half-life, resulting in enhanced analgesia. Penetration at the BBB was decreased, due to the conjugated compound enhanced hydropholicity, however the PEGylation of DPDPE did reduce efflux out of the brain by reducing DPDPE affinity for P-glycoprotein.

Peroxovanadium complexes have been identified as potent insulin mimetic agents and thus have been proposed for the treatment of diabetes. However, due to their nonselective inhibition of protein tyrosine phosphatases, these complexes affect a number of intracellular signaling pathways, both related and unrelated to insulin mimesis, jeopardizing their potential use as therapeutic agents. / Cytotoxic effects of three monoperoxovanadium complexes were studied with the hopes of establishing rules for determining cytotoxicity. MpV[pic] and mpV[dpc] displayed similar toxicity profiles and lead to the activation of members of the Mitogen Activated Protein Kinases (MAPK) superfamily in a dose- and time-dependent manner. On the other hand, mpV[NTA] appeared less toxic than the other two complexes studied, and accordingly, lead to a lower level, if at all, of MAPK phosphorylation. These results, although not conclusive, imply the participation of MAPKs in cell death. Cell death caused by these complexes presented features of both necrosis and apoptosis. / The second objective of this research was to further characterize the modulation of the ERK signaling cascade by the most commonly used peroxovanadium complex, bpV[phen] in a variety of different cellular contexts. It was discovered that signaling through a receptor with tyrosine kinase activity (RTK) coupled with cellular differentiation, as exemplified by the use of NGF and FGF, leads to ERK phosphorylation by 10 muM bpV[phen] for 1 hour that can be almost completely abolished by use of PD98059, an inhibitor of MEK. In contrast, non-differentiated cells, cells differentiated with a cyclic AMP analogue and PC12 cells treated with EGF appear to signal mostly through a non-PD98059-inhibitable pathway, when stimulated with the same dose of bpV[phen] for one hour.

No description available.