The pharmacology of CGRP-responsive receptors in cultured and transfected cells

Historically, CGRP receptors have been classified as CGRP(1) or CGRP(2) subtypes, chiefly depending on their affinity for the antagonist CGRP(8-37). It has been shown that the complex between calcitonin receptor-like receptor (CRLR or CL) and receptor activity modifying protein (RAMP) 1 provides a molecular correlate for the CGRP(1) receptor; however this does not explain the range of affinities seen for CGRP(8-37) in isolated tissues. It is suggested that these may largely be explained by a combination of methodological factors and CGRP-responsive receptors generated by CL and RAMP2 or RAMP3 and complexes of RAMPs with the calcitonin receptor.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

Mechanisms of ion channel activation in primary cultured and clonal cell lines

The effects of hypotonic shock upon membrane C1 permeability of ROS 17/2.8 osteoblast-like cells was investigated using the patch-clamp technique. Hypotonic shock produced cell swelling that was accompanied by large amplitude, outwardly rectifying, currents that were active across the entire physiological range of membrane potentials (-80 to +100 mV). At strong depolarisations (> +50 mV) the currents exhibited time-dependent inactivation that followed a monoexponential time course. The currents were anion selective and exhibited a selectivity sequence of SCN- > I > Br- > Cl- > F- > gluconate. Current activation was unaffected by inhibitors of protein kinase (A (H-89) and tyrosine kinase (tyrphostin A25), and could not be mimicked by elevation of intracellular Ca2+ or activation of protein kinase C. Similarly, disruption of actin filaments by dihydrocytochalsin B, or generation of membrane tension by dipyridamole failed to elicit significant increases in cell chloride permeability. The mechanism of current activation is as yet undetermined. The currents were effectively inhibited by the chloride channel inhibitors NPPB and DIDS but resistant to DPC. A Cl- conductance with similar characteristics was found to be present in mouse primary cultured calvarial osteoblasts. The volume-sensitive Cl- current in ROS 17/2.8 cells was inhibited by arachidonic acid in two distinct phases. A rapid block that developed within 10 s, preceding a slower developing inhibitory phase that occurred approximately 90 s after onset of arachidonate superfusion. Arachidonic acid also induced kinetic modifications of the current which were evident as an acceleration of the time-dependent· inactivation exhibited at depolarised potentials. Inhibitors of cyclo-oxygenases, lipoxygenases and cytochrome P-4S0 were ineffectual against arachidonic acid's effects sugtgesting that arachidonic acid may elicit it's effects directly. Measurements of cell volume under hypotonic conditions showed that ROS 17/2,8 cells could effectively regulate their volume, However, effective inhibitors of the volume-sensitive CI" current drastically impaired this response suggesting that physiologically this current may have a vital role in cell volume regulation, In L6 skeletal myocytes, vasopressin was found to rapidiy hyperpolarise cells. This appears to occur as the result of activation of Ca2+ -sensitive K+ channels in a process dependent upon the presence of extracellular Ca2+.

Tissue engineering of co-cultured skin substitutes using biocomposite membranes based on collagen and polycaprolactone

The preparation and characterisation of collagen: PCL, gelatin: PCL and gelatin/collagen:PCL biocomposites for manufacture of tissue engineered skin substitutes are reported. Films of collagen: PLC, gelatin: PCL (1:4, 1:8 and 1:20 w/w) and gelatin/collagen:PCL (1:8 and 1:20 w/w) biocomposites were prepared by impregnation of lyophilised collagen and/or gelatin mats by PCL solutions followed by solvent evaporation. In vitro assays of total protein release of collagen:PCL and gelatin: PCL biocomposite films revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the biocomposite samples that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. Good compatibility of all biocomposite groups was proven by interaction with 3T3 fibroblasts, normal human epidermal keratinocytes (NHEK), and primary human epidermal keratinocytes (PHEK) and dermal fibroblasts (PHDF) in vitro respectively. The 1:20 collagen: PCL materials exhibiting good cell growth curves and mechanical characteristics were selected for engineering of skin substitutes in this work. The tissue-engineered skin model based on single-donor PHEK and PHDF with differentiated confluent epidermal layer and fibrous porous dermal layer was then developed successfully in vitro proven by SEM and immunohistochemistry assay. The following in vivo animal study on athymic mice revealed early complete wound healing in 10 days and good integration of co-cultured skin substitutes with adjacent mice skin structures. Thus the co-cultured skin substitutes based on 1:20 collagen: PCL biocomposite membranes was proven in principle. The approach to skin modelling reported here may find application in wound treatment, gene therapy and screening of new pharmaceuticals.

Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

Insulin release from cultured B-cells

Established RlNm5F and lN111 R1 and newly available HlT-T15 and UMR 407/3 B-cell lines have been successfully maintained in vitro. With the exclusion of UMR 407/3 cells, all lines were continuously propagable. Doubling times and plating efficiencies for HlT-T15, RlNm5F, lN111 R1 and UMR 407/3 cells were 20 hours and 85%, 31 hours and 76%, 24 hours and 80% and 38 hours and 94% respectively. All the cell lines were anchorage dependent, but only UMR 407/3 cells grew to confluence. Only HlT-T15 and UMR 407/3 cells produced a true insulin response to glucose but glucose markedly increased the rate of D-[U14C]glucose oxidation by all the cell lines. Glucose induced insulin release from HlT-T15 cells was biphasic with an exaggerated first phase. Insulin release from HlT-T15, RlNm5F and IN111 R1 cells was stimulated by amino acids and sulphonylureas. Glucagon stimulated insulin release from HlT-T15 and RlNm5F cells while somatostatin and pancreatic polypeptide inhibited release. These observations suggest that net insulin release from the whole islet may be the result of significant paracrine interaction. HlT-T15 and RlNm5F cell insulin release was stimulated by forskolin and inhibited by imidazole. Ca2+ channel blockade and calmodulin inhibition suppressed insulin release from HlT-T15, RlNm5F and IN111 R1 cells. In addition phorbol esters stimulated insulin release from RlNm5F cells. These data implicate cAMP, Ca2+ and protein kinase-C in the regulation of insulin release from cultured B-cells. Acetylcholine increased insulin release from HlT-T15 and RlNm5F cells. Inhibition of the response by atropine confirmed the involvement of muscarinic receptors. HlT-T15 cell insulin release was also inhibited by adrenaline. These observations suggest a possible role for the autonomic nervous system in the modulation of insulin release. Preliminary studies with a human insulinoma maintained in monolayer culture have demonstrated a limited life span of some seven weeks, a continuous low level of insulin release but no insulin response to glucose challenge.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Absorption and metabolism of chlorogenic acids in cultured gastric epithelial monolayers

Gastric absorption of feruloylquinic acid and di-O-caffeoylquinic acid analogs has never been investigated despite their potential contribution to the proposed beneficial health effects leading to reduced risk of type 2 diabetes. Using a cultured gastric epithelial model, with an acidic apical pH, the relative permeability coefficients (P(app)) and metabolic fate of a series of chlorogenic acids (CGAs) were investigated. Mechanistic studies were performed in the apical to basal direction and demonstrated differential rates of absorption for different CGA subgroups. For the first time, we show intact absorption of feruloylquinic acids and caffeoylquinic acid lactones across the gastric epithelium (P(app) ~ 0.2 cm/s). Transport seemed to be mainly by passive diffusion, because good linearity was observed over the incubation period and test concentrations, and we speculate that a potential carrier-mediated component may be involved in uptake of certain 4-acyl CGA isomers. In contrast, absorption of intact di-O-caffeoylquinic acids was rapid (P(app) ~ 2-10 cm/s) but nonlinear with respect to time and concentration dependence, which was potentially limited by interaction with an efflux transporter and/or pH gradient dependence. For the first time, methylation is shown in gastric mucosa. Furthermore, isoferulic acid, dimethoxycinnamic acid, and ferulic acid were identified as novel gastric metabolites of CGA biotransformation. We propose that the stomach is the first location for the release of hydroxycinnamic acids, which could explain their early detection after coffee consumption.

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals. © 2005 Elsevier Inc. All rights reserved.

High permeability of the anionic form restricts accumulation of indomethacin by cultured gastric surface epithelial cells exposed to low apical pH

The 'ion-trapping' hypothesis suggests that the intracellular concentration of acidic non-steroidal anti-inflammatory drugs in gastric epithelial cells could be much higher than in the gastric lumen, and that such accumulation could contribute to their gastrotoxicity. Our aim was to examine the effect of the pH of the apical medium on the apical to basal transfer of the acidic drug indomethacin (pK a 4.5) across a gastric mucous epithelial cell monolayer, and to determine whether indomethacin accumulated in cells exposed to a low apical pH. Guinea-pig gastric mucous epithelial cells were grown on porous membrane culture inserts (Transwells®) for 72 h. Transfer and accumulation of [ 14C] indomethacin were assessed by scintillation counting. Transfer of [ 3H]mannitol and measurement of trans-epithelial electrical resistance were used to assess integrity of the monolayer. Distribution of [ 14C] urea was used to estimate the intracellular volume of the monolayer. The monolayer was not disrupted by exposure of the apical face to media of pH ≥ 3, or by indomethacin. Transfer of indomethacin (12 μM) to the basal medium increased with decreasing apical medium pH. The apparent permeability of the undissociated acid was estimated to be five times that of the anion. The intracellular concentration of indomethacin was respectively 5.3, 4.1 and 4.3 times that in the apical medium at pH 5.5, 4.5 and 3.0. In conclusion, this study represents the first direct demonstration that indomethacin accumulates in gastric epithelial cells exposed to low apical pH. However, accumulation of indomethacin was moderate and the predictions of the ion-trapping hypothesis were not met, probably due to the substantial permeability of anionic indomethacin across membranes. © 2006 Elsevier B.V. All rights reserved.