Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) is tightly regulated by cAMP-dependent phosphorylation, but the protein phosphatases (PP) that dephosphorylate and inactivate CFTR remain poorly understood. Differential regulation of single CFTR channels by protein phosphatase 2C (PP2C), protein phosphatase 2A (PP2A) and other phosphatases has been reported. The results suggest multiple protein phosphatases are required for complete CFTR deactivation, and that PP2C may be the primary phosphatase regulating CFTR in human airway and colonic epithelia, two major sites of disease. / The goal of this project was to identify PP2C isoforms that regulate CFTR. Six isoforms of PP2C reported previously in mouse were recently recloned in this laboratory with epitope tags, and their interaction with CFTR was assessed by co-immunoprecipitation. Preliminary results indicated that MB2.1, one of the PP2Cbeta isoforms, and MA3, one of the PP2Calpha isoforms, interact with CFTR. To overexpress these phosphatases, I developed two inducible systems in which MB2.1 or MA3 gene expression is controlled by a promoter that is activated upon treatment of cells with ponasterone A, an ecdysone analogue. Although specific PP2C inhibitors are not presently available, mutagenesis and enzyme assays have identified residues that are essential for its enzymatic activity. I made PP2Calpha and PP2Cbeta mutants which are predicted to act as dominant negative inhibitors, and have stably transfected them using the inducible gene expression system. Furthermore, I showed that MB2.1 and MA3 are functionally expressed in the inducible expression system and that site-directed mutagenesis inactivated their phosphatase activity as assayed using phosphatase p-nitrophenyl phosphate (pNPP) as a substrate. Baby Hamster Kidney (BHK) cells stably expressing CFTR + inducible MB2.1, MA3 or their dominant negative mutants, are presently being used in iodide efflux assays to study the downregulation of CFTR by MB2.1 and MA3. Based on results, we exclude PP2C beta regulating CFTR. However, if PP2C alpha is the phosphatase which dephosphorylates CFTR can not be tested because of iodide loading problem.

This work focused on the natural development of neurotachykinin-1 (NK-1) receptors in the porcine brainstem during postnatal development (using 4 ages vs adult), and on alterations in these receptors after single and six-daily repeated clustered hypoxia (using young and older piglets). NK-1 receptor localization and densities were determined by quantitative autoradiography, using mono-iodinated Bolton-Hunter substance P ([125I]-BHSP). Slide-mounted brainstem sections, incubated in [125I]-BHSP and then exposed to film, have shown [125I]-BHSP binding throughout many brainstem nuclei and tracts, including the ambigual/periambigual (nAmb), dorsal motor vagal (dmnv), hypoglossal (nHyp), lateral reticular (nRL), gigantocellular (nGC), solitary tract (nTS), medial parabrachial (nPBM), and raphe obscurus (nROb) nuclei. NK-1 receptor densities decreased with age. As compared to normoxia, NK-1 receptor densities increased significantly after the 6-daily hypoxia protocol in dmnv, nHyp, nRL, nROb, and nTS of both young and older age groups. This increase may represent receptor upregulation as an adaptation to repeated hypoxia.

Stimulating a rabbit ventricular cell with a train of current pulses at a long basic cycle length (BCL) and high pulse amplitude (PA) leads to a 1:1 rhythm, where each injected pulse generates an action potential. With a fixed BCL (BCL = 300 ms at 34--36°C and BCL = 1000 ms at 22--24°C), as PA is decreased, there is a transition from 1:1 rhythm to Wenckebach rhythms. These rhythms are characterized by a beat-to-beat increment in latency and action potential duration (APD) culminating in a skipped beat. Using voltage and action potential clamp, as well as a specific channel blocker, I show that the transient outward current (Ito) is implicated in the generation of these APD increments, and is responsible for the periodicity of these rhythms. When PA is raised, a direct transition from 1:1 to 2:1 rhythm can be observed by varying BCL. With the use of an ionic model, hysteresis between 1:1 and 2:1 rhythms is shown and compared to similar hysteresis observed previously in experiments with rabbit ventricular cells. When the BCL is within the hysteresis range, the addition of stimulus pulses flips 1:1 to 2:1 rhythm and vice versa, providing evidence for bistability. With the ionic model, the substraction of a single pulse also flipped 1:1 to 2:1 rhythm. Iteration of a finite-difference equation, derived using the steady-state APD restitution curve, predicts the direct {1:1 ↔ 2:1} transition, as well as bistability, in both the experimental and modeling work. Finally, I show in an ionic model that the transition from 1:1 to 2:1 rhythms: (1) is interrupted by Wenckebach rhythms at low PAs; (2) is direct without hysteresis at one specific higher PA value; (3) is direct with hystersis at still higher PAs. While iteration of the APD restitution curve predicted {1:1 ↔ 2:1} bistability at higher PA, iteration of the latency restitution curve predicted Wenckebach rhythms at lower PA, confirming the importance of latency for the existence of Wenckebach rhythms

The aim of the work presented here is to evaluate the effects of cannabinoids in three animal models of pain: acute, tonic and chronic. The tail flick test (acute pain) was used to test the effect of the cannabinoid agonist, WIN 55,212--2, on tail withdrawal latency from a noxious radiant heat source. It was also tested on the allodynia induced by either endogenous release or exogenous administration of substance P. WIN 55,212--2 was antinociceptive in this test, and blocked the substance P-induced allodynia, suggesting a post-synaptic site of action. The formalin test (tonic pain) was used to test the effects of the endogenous cannabinoid agonist, anandamide and the cannabinoid receptor antagonist AM 281. Anandamide was antinociceptive (with a short duration of action), and AM 281 was pronociceptive. When administered concomitantly, AM 281 blocked the effects of anandamide. When given alone and in the absence of a noxious stimulus, AM 281 was without effect. (Abstract shortened by UMI.)

This work has focused on understanding in vivo aspects of ASP metabolism particularly postprandially. I showed that ASP was produced directly from human adipose tissue, a process that increased postprandially and was correlated with increased plasma triglyceride clearance and increased fatty acid incorporation into adipose tissue "FIAT". The target of ASP is adipose tissue. Binding studies in freshly obtained adipose tissue demonstrated specific binding to high affinity receptor sites. ASP binding to subcutaneous tissue from obese females demonstrated the greatest binding and highest receptor affinity. In contrast omental tissue particularly from males showed the lowest specific binding and affinity. This suggests an important role for ASP in maintaining regional adipose tissue mass in females and males, hence providing possible explanations for the metabolic complications seen in abdominal obesity. / The strongest evidence for a physiological role of ASP on triglyceride clearance for ASP in vivo was obtained when exogenous intraperitonial hASP was administered to genetically obese mice. Normolipidemic ob /ob mice demonstrated accelerated postprandial TG clearance in the presence of ASP. The effect in the hyperlipidemic: db/db mice, however was markedly greater (2 to 8 times). These findings strongly support the hypothesis that ASP is an important factor in postprandial lipid metabolism and may be a significant factor in determining the pathophysiology of obesity and related dyslipidemias.

Under normal conditions the sinoatrial node serves as the pacemaker of the heart. However, under disease states other pacemaker sites can emerge which compete with the sinoatrial node or with each other. This thesis describes theoretical and experimental studies of pacemaker initiation and interaction. The first aspect of this thesis deals with an arrhythmia called modulated parasystole, which is generated by the interaction between the sinus pacemaker and an ectopic pacemaking focus. A mathematical model was developed to study the dynamics of modulated parasystole using discontinuous circle maps. The mathematical model displayed banded chaos, characterized by zero rotation interval width in the presence of a positive Lyapunov exponent. Banded chaos in the parasystole map produces rhythms characteristic of those found clinically. The second aspect of the thesis deals with spontaneous pacemaker activity and interaction using optical mapping techniques and mathematical models. A macroscopic imaging system was designed and constructed that records fluorescent signals from thin preparations over large areas (1 cm2 ) for long time periods (>30 minutes). Rotating waves (rotors) of cellular activity were observed by mapping calcium in nonconfluent cultures of embryonic chick heart cells. Unlike previous observations of rotors or spiral waves in other systems, the rotors did not persist but exhibited a repetitive pattern of spontaneous onset and offset leading to a bursting rhythm. Similar dynamics were observed in simple excitable media models that incorporated spontaneous initiation of activity and a decrease of excitability as a consequence of rapid activity (fatigue). These results provide a mechanism for bursting dynamics in normal and pathological processes. Activation maps were also obtained from the rabbit atrioventricular (AV) node, a small region of the heart with specialized pacemaking and conducting properties. This work determined sites of delay and spontane

Neuronal nicotinic acetylcholine receptors (nAChRs) are wide spread in the nervous system. Ample evidence indicates that many central nAChRs are located at the nerve terminals, where they act to facilitate neurotransmitter release; however, little is known about how these receptors carry out their function. The focus of my study is to understand the mechanism(s) that underlie the function of neuronal nAChRs. Neuronal nAChRs conduct inward current at negative membrane potentials, but conduct little or no outward current at positive membrane potentials, a process known as inward rectification. Inward rectification prevents the ACh-evoked conductance increase from short-circuiting the action potential at the nerve terminal, thereby ensuring optimal depolarization of the terminal and effective neurotransmitter release. Using the outside-out single channel patch-clamp technique, I demonstrate that intracellular polyamines block neuronal nAChRs with high affinity and in a voltage dependent manner; this is true for native nAChRs expressed by sympathetic neurons as well as recombinant alpha3beta4, alpha4beta2 nAChRs expressed in Xenopus oocytes. Given the physiological concentrations of polyamines inside cells, this block can fully account for the strong macroscopic inward rectification. Furthermore, using a combined approach of site-directed mutagenesis and electrophysiology, I show that the negatively charged residues at the cytoplasmic mouth of the pore (known as the intermediate ring) mediate the interaction of intracellular polyamines with the receptor; partial removal of these residues abolishes the strong inward rectification. Interestingly, I show that the intermediate ring influences the permeation of calcium through the receptor, indicating that a molecular link exists between calcium permeability and inward rectification of neuronal nAChRs. My experiments also show that extracellular polyamines and a polyamine-related toxin, Joro spider toxin, block neuronal n

The excitability properties of multicellular and single cellular cardiac pacemaking tissues was examined using mathematical, electrophysiological and molecular biological techniques. Particular attention was focused on identifying the different ionic channels that control the excitability of a physiological pacemaker, the rabbit atrioventricular node. The first part of the study centers on the excitability and rhythmicity of rabbit atrioventricular cell clusters and embryonic chick heart cell aggregates to compare cardiac pacemaking tissues showing slow- and fast-type action potentials. Subsequent experiments examined the membrane excitability properties of single isolated atrioventricular nodal cells and characterized the principle ionic currents that govern excitability. One of these channels that is directly gated by cyclic nucleotides, the "pacemaker" channel (If), was found to be non-uniformly expressed in different populations of AV nodal cells. The final part of the thesis involved a molecular characterization of cyclic nucleotide-gated ion channel subunits expressed in cardiac tissue during embryonic development. The relationship of these channels to the cardiac "pacemaker" channel (If) and their possible role in controlling cardiac excitability were considered.

No description available.

This study has focussed on the formation and survival of acetylcholine receptor (AChR) clusters at neuromuscular synapses formed in culture between myotomal muscle cells and spinal cord neurons derived from embryos of Xenopus laevis. AChRs were labelled with tetramethylrhodamine-conjugated $ alpha$-bungarotoxin so that the neurite associated receptor patches (NARPs) on the muscle cells could be viewed by fluorescence microscopy. Reduced fluorescence excitation was used in combination with a low light level TV camera and a computer based image calculator to make daily observations on all NARPs. Observations suggest the following conclusions. Neurons retain the capacity to trigger NARP formation as long as they continue to grow. Changes in NARP shape as well as decreases (and increases) in NARP size can occur even in the absence of competitive interactions between neurons and these changes are locally regulated along the contact. The capacity of neurons to maintain NARPs in more proximal portions of their neuritic arbor persists even as growing distal portions continue to induce the formation of new NARPs.