Delta opioid receptor agonists have been proposed as a new generation of opioids analgesics. They have been implicated in antinociception in the spinal cord in acute and chronic pain models. Yet the precise means by which this role is achieved is still unclear. The first aim of this study was to determine whether delta opioid receptors could modulate the action of pro-nociceptive transmitters in the spinal cord using reflex studies. The second aim of this study was to examine the effect of a delta-1 opioid receptor agonist on excitatory amino acid evoked responses and synaptically elicited responses of rat single spinal dorsal horn neurons to natural stimulation of peripheral receptive fields in electrophysiological experiments. / Our findings in the first part of the study indicate that the mechanism of antinociception of delta opioids at the spinal level may include a selective blockade of the action of some of pro-nociceptive transmitters. / In the second part of the study, the preferential inhibition of DPDPE on the after discharge induced by noxious stimulation and NMDA induced excitation suggests that delta opioid receptor agonists may have the potential to treat chronic pain. (Abstract shortened by UMI.)

Iron (Fe) plays an essential role in numerous metabolic processes. The transferrin receptor (TfR) is a cell membrane-associated protein that serves as a gatekeeper in regulating cellular uptake of Fe from transferrin (Tf) by TfR-mediated endocytosis. TfRs are expressed ubiquitously, but their highest levels of expression are on immature erythroid cells (which are the most avid consumers of Fe in the organism) and on rapidly dividing cells, both normal and malignant. In proliferating non-erythroid cells TfR expression is feedback inhibited by Fe through the interaction of specific binding proteins, iron regulatory proteins (IRPs), with iron responsive elements (IREs) in the 3 ' untranslated region (UTR) of TfR mRNA. However, results from our lab indicate that in differentiating murine erythroleukemia (MEL) cells, TfR expression responds only slightly to an increase in intracellular Fe. An explanation for this is lacking. Importantly, a second TfR (TfR2) has recently been cloned in humans. TfR2 is highly homologous with the classical receptor, TfR1 (except for the lack of IREs in the 3' UTR), and seems to have a similar function, but shows different tissue distribution. It has been speculated that TfR2 is expressed in erythroid cells since its message is abundant in K562 cells, a human cell line that can differentiate along an erythroid lineage, and can be induced to synthesize hemoglobin. Thus, it is important to determine whether TfR2 is expressed in erythroid cells, since this could possibly explain differences in TfR regulation in erythroid vs. non-erythroid cells. In this study, I have cloned mouse TfR2 (mTfR2) by RACE (rapid amplification of cDNA ends) and sequenced it. The sequence analysis shows approximately 50% and 82% homology to mouse TfR1 and human TfR2, respectively. Tissue distribution of mTfR2, based on Northern blot analysis indicated that, in the mouse, TfR2 mRNA was predominantly expressed in the liver and its level increased about 2 fold after a

Brief episodes of ischemia followed by reperfusion render the heart more resistant to injury from a subsequent prolonged ischemia. This endogenous and highly protective phenomenon is known as ischemic preconditioning. Although the phenomenon has been demonstrated virtually in all species tested including nun, the mechanism of ischemic preconditioning is incompletely understood. Early studies showed that both adenosine A1 receptors and K ATP channels appeared to mediate the effects of preconditioning in most species, but not in the rat. Therefore, we first investigated the signal transduction mechanisms involved in ischemic preconditioning against post-ischemic contractile dysfunction in isolated Langendorff-perfused rat hearts. We found that ischemic preconditioning in the rat heart is due to stimulation of alpha1B-adrenoceptors by release of endogenous catecholamines, resulting in acfivation of a pertussis toxin-sensitive guanine nucleotide binding (G) protein which enhances protein kinase C (PKC) activity. The results support the hypothesis that G protein-dependent PKC activation may be the common mechanism in signal transduction pathways of ischemic preconditioning among species. Accumulating evidence suggests that ATP-sensitive K+ channel (KATP channel) activation plays an important protective role in ischemic preconditioning. We hypothesized that PKC stimulation in ischemic preconditioning may produce cardioprotection by modulating KATP channel function, and studied further the modulation of the KATP channel by PKC-mediated phosphorylation, in rabbit and human ventricular myocytes, using voltage clamp techniques. We found that PKC activation stimulates KATP-induced opening at reduced intracellular ATP concentrations by reducing KATP channel sensitivity to intracellular ATP. In order to determine whether membrane-bound PKC could explain previously-described adenosine-induced KATP channel activation in excised membrane patches, we studied KATP channel function

This thesis is about the development and evaluation of a standardized method to measure the crural diaphragm electromyogram (EMG) in humans, with an esophageal electrode. In order for the diaphragm EMG to be physiologically relevant, its measurement and analysis require objective control of the disturbances and filter effects which can influence the sigma. One issue of importance is the maintenance of diaphragm-to-electrode positioning throughout the inspiration. In the present work, we describe a cross-correlation algorithm by which the position of the diaphragm along a multiple army esophageal electrode can be determined at any instant during a breath, and a second algorithm, the "double subtraction technique", which further minimizes the detrimental effects of diaphragm movement. The double subtraction technique also results in the improvement of the diaphragm EMG signal to noise ratio by 2 dB. By implementing these algorithms, we demonstrate in healthy subjects that there is no artifactual influence of lung volume/chest wall configuration on the diaphragm EMG frequency content nor on the diaphragm EMG signal strength during voluntary isometric contractions of the diaphragm. This is in contrast to the electrically elicited diaphragm compound muscle action potentials which are severely influenced by changes in lung volume. We also show that the volume-activation relationship of the diaphragm (required change in activation for changes in lung volume at a given tension) is directly related to the length-tension properties of the muscle. During dynamic, voluntary breathing maneuvers, we could find no evidence for an increase in diaphragm EMG signal strength when inspirations from functional residual capacity to total lung capacity (TLC) were performed at increasing inspiratory flow rates (velocities of shortening) up to 1.4 l/s. To account for anatomical and physiological differences between subjects, we demonstrate that the diaphragm EMG signal strength can be no

Studies were designed to investigate the expression and regulation of interstitial collagenase (MMP-1) during endochondral ossification in fetal bovine growth plate chondrocytes in vitro. Fetal bovine growth plate chondrocytes were separated into subpopulations representing different maturational stages by using a Percoll discontinuous gradient. Gene expression and synthesis of MMP-1 in these chondrocyte subpopulations were followed in serum-free cultures as they matured from the prehypertrophic to hypertrophic phenotype. However, MMP-1 mRNA expression and protein were undetectable. This expression was also undetectable in interleukin 1alpha (IL-1alpha) stimulated growth plate chondrocytes. MMP-1 mRNA and protein were however observed in IL-1alpha stimulated bovine fetal skin fibroblasts. Although MMP-1 was absent in growth plate chondrocytes, a collagenolytic activity was detectable in culture medium by using 14C labeled type II collagen. It was only seen when the chondrocytes were hypertrophic. By using the polymerase chain reaction (PCR), employing conserved sequences found in mammalian collagenase-3 (MMP-13), a novel bovine collagenase, was cloned. The cDNA and predicted amino acid sequence shared a 92% and 90% identity to the human MMP-13 cDNA and amino acid sequence respectively. MMP-13 mRNA and protein were found only when the chondrocytes became hypertrophic. The role of MMP-13 during the maturation of these fetal growth plate chondrocytes was further investigated by using a preferential carboxylate inhibitor of collagenase-3. This inhibitor reduced the generation of denatured type II collagen in chondrocyte cell layers and proteoglycan release into conditioned medium in a dose dependent manner. Also, types II and X collagen synthesis and matrix calcification were inhibited. These studies indicate that MMP-13 may play a very important role during the maturation of fetal growth plate chondrocyte in vitro and during endochondral ossification and that cellul

Fibrinogen (Fg) mediates platelet aggregation and adhesion to artificial surfaces by interacting with its receptor, glycoprotein IIb and IIIa complex (GPIIbIIIa, or integrin alphaIIbbeta 3), on the platelet membrane. Considerable evidence has demonstrated that the (H12) on the gamma chain carboxyl terminus is required for the binding of Fg from solution to activated platelet GPIIbIIIa, while the RGD sites, the universal integrin recognition domain on adhesive ligands, are not involved. In this study, using recombinant Fg, well-defined Fg plasmin digestion fragments, and specific monoclonal anit-Fg antibodies, we demonstrated that the same sequence, the H12, or more precisely, the AGDV on the extreme carboxyl terminus of the gamma chain (gamma408--411), is also required for platelet-bound Fg to support platelet aggregation (crosslinking), thus experimentally verifying the "two sticky ends" theory of Fg-mediated platelet aggregation The RGD-containing domains on the Aalpha chains are not involved in aggregation. The AGDV sequence on the gamma chain carboxyl terminus is also necessary and sufficient for activated platelets to adhere to surface-adsorbed Fg, while the RGD sequences we similarly not required. A receptor induced binding site (RIBS), the Fg RIBS-I site (gamma373--385), on Fg either bound to its GPIIbIIIa-receptor or on a surface, is not directly involved in interactions between platelet GPIIbIIIa and immobilized Fg. The inhibitory effects of the anti-Fg-RIBS-I antibody are due to steric hindrance of the accessibility of the AGDV site to platelet GPIIbIIIa. Thus, the extreme carboxyl terminus of the gamma chain is the only site in both fluid and solid phase Fg that is involved in platelet GPIIbIIIa-Fg interactions. / Though resting platelets are able to adhere to surface-bound Fg, this adhesion efficiency is much lower than that of the adhesion of the activated platelets. The adhesion efficiency of both resting and activated platelets to surface-adsorbed Fg decreases with increasing shear rate from 100 s -1 to 2,000 s-1. However, the decrease of the adhesion efficiency of the resting platelets is more marked than the decrease of the adhesion efficiency of the activated ones. Thus, the higher the shear rates, the larger the difference in the adhesion efficiencies between resting and activated platelets. However, due to the higher collision frequencies at higher shear rates, the adhesion of resting platelets was maintained at a similar level from shear rates of 300--2,000 s-1, while the adhesion of activated platelets kept increasing from 100 s -1 to 2,000 s-1. These data indicate that platelet activation is an efficient regulation pathway for platelet adhesion to surfaces.

Circadian rhythms, ∼24-h oscillations in physiological variables, are pervasive throughout nature. Synchronization of these oscillations to the 24-h day enables organisms to take advantage of environmental cycles. In mammals, a circadian pacemaker located in the hypothalamus coordinates the overt rhythms. / The present study investigates aspects of the 24-h organization of breathing, and its chemical control, and of thermoregulation in mammals. Pulmonary ventilation (V˙E), oxygen consumption ( V&d2;O2 ), body temperature (Tb), and locomotor activity were monitored by non-invasive means, in freely-moving male adult rats. Rats are nocturnal, and it is well known that their Tb, activity and metabolic rate are highest during the dark hours of the day. / Hypoxia inhibits thermogenesis, and the Tb and V&d2;O2 circadian patterns are contributed to by changes in thermogenesis, implying that hypoxia blunts the daily oscillations of these variables. Indeed, the amplitude of both oscillations was smaller, due to a decrease in the dark phase values. Evidence supports an action of hypoxia on the hypothalamic thermoregulatory mechanisms, rather than on the clock itself. / Metabolism is well known to be a major determinant of V˙E, and of the V˙E response to changes in inspired gases. Using a custom-designed system to monitor breathing continuously, during air breathing, V˙ E was found to oscillate, with higher values during the dark compared to the light hours of the day; these changes were almost in proportion to those of V&d2;O2 , and did not depend on those of activity. / The depressant effect of hypoxia on the high values of the V&d2;O2 oscillation predict that the hypoxic V˙E response would be blunted at this time. Indeed, the response was lower during the dark compared to the light hours; however, the daily changes in the V˙E response were in proportion to those of V&d2;O2 , such that the hyperventilatory response (% increase in V˙ E/ V&d2;O2 ) was similar throughout the day. The V˙E/ V&d2;O2 response was also similar throughout the day in hypercapnia, even though the metabolic response to hypercapnia differed from that in hypoxia. / Globally taken, these results indicate that (1) breathing and its control mechanisms accompany the daily oscillations of many physiological variables, and (2) the advantages of a biological clock do not compromise the adequacy of the hyperventilatory responses to chemical challenges.

The effects of activation of protein kinase C (PKC) on intermediate filaments (IFs) of different cell types were investigated. In primary cultures of murine or rabbit spinal cord, or human fibroblasts, brief exposure to activators of PKC led to the following changes in IFs: (1) Disassembly of GFAP-filaments in astrocytes and of vimentin in fibroblasts and fragmentation of the neurofilament (NF) network in neuronal cell bodies (occurred within 30 minutes). The distribution of PKC isoforms was investigated in these cell types and no IF-associated isoform was found. Disassembly of IFs may be explained by direct phosphorylation of N-terminal domains of IFs by PKC. (2) Activation of PKC led also to changes similar to those occurring in various motor neuron diseases: Focal swellings of proximal neurites of motor neurons, but not other types of neurons (within hours), and hyperphosphorylation of C-terminal domains of NF proteins (persisting over days). NF phosphorylation was determined by increased immunoreactivity with antibodies SMI31 and SMI34 recognizing NF proteins (NF-M & NF-H) when C-terminal KSP repeat domains are in intermediate and hyperphosphorylated states, respectively. Increased SMI34 labeling was prevented by pretreatment with the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), but not by the AMPA/kainate antagonist, CNQX, or the metabotropic glutamate receptor antagonist, MCPG, indicating a cooperative effect of NMDA receptor activation in PKC-induced NF hyperphosphorylation. Since PKC does not phosphorylate C-terminal domains of NF proteins, involvement of other kinases was investigated. Hyperphosphorylation of NFs induced by PKC was prevented by the specific Ca$ sp{2+}$/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62, but not by its analogue, KN-04. The following mechanism is proposed for NF hyperphosphorylation in motor neuron diseases: Activation of PKC, as a nonspecific response to injury, acts cooperati / The results of the present thesis together demonstrate multiple effects of PKC activation on modulation of IFs that might contribute to dysfunction of cells in certain diseases.

This thesis focuses on the functional role of monosialoganglioside (GM1) in neuronal activity and synaptic transmission of rat hippocampal slices. The slices were placed in an interface recording chamber and constantly superfused with oxygenated saline at 33$ sp circ$. Both extracellular and intracellular (current- and voltage-clamp) recording techniques were used to measure the field potentials and postsynaptic responses respectively. Our findings indicated that GM1 induces a small inward current in CA1 pyramidal neurons, depresses their high voltage activated (HVA) Ca$ sp{2+}$ currents, and selectively facilitates the excitatory synaptic inputs while reducing the inhibitory ones. / The most probable mechanism underlying the selective enhancement of excitatory inputs by GM1 is an increase in glutamate release, since the amplitudes and frequency of spontaneous miniature postsynaptic responses, recorded either in the presence or absence of presynaptic cell firing, were cosistently increased. When L-glutamate was applied iontophoretically in the dendritic region, GM1 transiently potentiated the postsynaptic glutamate currents, thus further indicating a GM1-induced enhancement of glutamatergic transmission. In contrast, both spontaneous and evoked inhibitory postsynaptic responses were suppressed by GM1. This effect is dependent on changes in excitatory inputs to inhibitory interneurons because in the presence of tetrodotoxin and/or kynurenic acid, GM1 did not alter the amplitude of the monosynaptic IPSPs or the frequency of spontaneous miniature IPSCs. / Both the GM1-induced inward current and the reduction of postsynaptic HVA Ca$ sp{2+}$ currents were antagonised by kynurenic acid, suggesting that these effects might be caused by glutamate receptor activation. By raising intraneuronal Ca$ sp{2+}$ concentration, the potentiated glutamate release would trigger Ca$ sp{2+}$-dependent Ca$ sp{2+}$ inactivation, and thus explain the reduction in HVA Ca$ sp{2+}$ currents. / In conclusion, most of the GM1 actions observed in this project can be explained on the basis of a GM1-induced facilitation of excitatory transmission, mediated especially via enhanced glutamate release.

L-histidine ammonia-lyase (histidase) was encapsulated within cellulose nitrate artificial cells, and its kinetic parameters were evaluated. Microencapsulated histidase had an apparent activity of approximately 50% of the activity of histidase in solution. Encapsulation did not alter the K$ sb{ rm M}$ of histidase. The K$ sb{ rm M}$ of histidase solution and the K$ sb{ rm M}$ apparent of microencapsulated histidase were both 20mM. Encapsulation of histidase resulted in increased stability of enzymatic activity of storage temperatures of 4$ sp circ$C and 37$ sp circ$C. At 37$ sp circ$C histidase solution reached 50% of its original activity after 9.5 days of storage, while microencapsulated histidase reached the same level after 15 days. At 4$ sp circ$C histidase solution had 63% of its original activity after 21 days of storage, while encapsulated histidase had 95%. In vitro experiments to evaluate the feasibility of microencapsulated histidase for possible experimental therapy in histidinemia were carried out. These experiments evaluated the effectiveness of encapsulated histidase in depleting histidine. Three different volume ratios of histidase loaded artificial cells to substrate solution were tested. A ratio of 1:100 allowed 25% histidine depletion after 120 hours. A 1:50 ratio allowed 35% histidine depletion after 72 hours. A 1:25 ratio allowed 40% histidine depletion after 24 hours.