For two decades, multiple observations of nonparallel pancreatic secretion, wherein digestive enzyme proportions change rapidly following various digestive stimuli, have conflicted with the concept of exocrine pancreatic homogeneity and the exocytosis model of parallel synthesis, transport and secretion of proteins. Evidence of pancreatic heterogeneity is presented, potentially resolving this longstanding controversy. Correlation and regression analysis simultaneously demonstrated exocytosis and nonparallel secretion, suggesting the existence of multiple heterogeneous exocytotic pathways. Next, heterogeneous prestored pancreatic protein sources were directly demonstrated using double isotopic labelling; temporal and secretagogue-specific regulation of the heterogeneous secretory sources was uncovered. Finally, specific enzyme proportions were linked to the heterogeneous sources by densitometric measurements of electrophoretic gels of secreted proteins. Thus, it appears that differential secretion from heterogeneous sources of prestored secretory proteins containing unique proportions of digestive enzymes is the basis of regulated nonparallel secretion in the exocrine pancreas.

This thesis investigates the effects of substance P on a spinal nociceptive reflex in the rat. Intrathecal administration of substance P and other analogous peptides to the L5 vertebral level produced a dose-dependent decrease in reaction time to tail withdrawal from a noxious radiant heat source. Similar administration of VIP, galanin, CGRP, angiotensin II, somatostatin and TRH failed to mimic these effects of substance P. As substance P was found to equally decrease the reaction time in spinal transected rats, it is concluded that this facilitation occurs via a spinal mechanism alone. / Administration of substance P at the lower thoracic level increased reaction time. Evidence is provided to suggest that this increase in reaction time may involve the release of an opioid peptide into the circulation from the adrenal medullae. / Noxious cutaneous stimulation to the tail of anaesthetized rat produced a facilitation of the tail flick reflex which was similar to that induced by exogenous administration of substance P. This decrease in reaction time was attenuated by intrathecal administration of a substance P antagonist. / These results support an earlier hypothesis made by others that substance P may be involved in the transmission of nociceptive information in the spinal cord.

The experimental work presented in this thesis explores, in anaesthetized and mid-collicular decerebrate, unanesthetized, cats, the properties of two components of the discharge of sympathetic nerves which are time-locked to the central respiratory cycle and are presumably generated within the central nervous system. One is the inspiration-synchronous burst, the other is a previously unknown late-expiratory burst. The properties of the inspiration-synchronous burst and its temporal relation to the phrenic nerve burst were studied under conditions in which the frequency of the latter was changed over a wide range by superior laryngeal nerve stimulation, by changes in ventilation frequency while the phrenic nerve burst was locked to the pump, and by hypocapnic hyperthermia. The data obtained are consistent with the hypothesis of a common rhythmic driver for phrenic motoneurons and sympathetic preganglionic neurons. Stimulation of low-threshold afferents in the superior laryngeal nerve selectively suppressed the phrenic burst together with the inspiration-synchronous sympathetic discharge and produced vasodilatation. The contribution of the inspiration-synchronous sympathetic discharge to neurogenic vasoconstriction was estimated, in the hindlimb of the cat, from the magnitude of the vasodilatation. A late-expiratory burst of sympathetic discharge was produced by systemic hypercapnia and by raising end-expiratory pressure to between 2 and 7 cmH$ sb2$O. Circumstantial evidence suggests this late-expiratory burst is due to input from late-expiratory neurons to sympathetic preganglionic neurons. As a background to the experimental data a survey is presented of present knowledge of the mechanisms providing mechanical and neural coupling between respiration and circulation. The functional significance of respiratory modulation of sympathetic activity is discussed.

This thesis focusses on the seizure-promoting effects of second messengers, in particular calcium and others activated by calcium in CA1 subfield of rat hippocampus in situ and in vitro. Interictal discharges were simulated in hippocampal slices using the low Ca$ sp{2+}$ model where synaptic transmission is blocked. The antiepileptic drug valproic acid (VPA), which is postulated to potentiate GABAergic responses, reduced the low Ca$ sp{2+}$ field bursts, thereby suggesting a non-synaptic mechanism of action of VPA. Extracellular and intracellular recordings from hippocampal neurones bathed in low Ca$ sp{2+}$ solution revealed that even though evoked transmitter release was blocked, small spontaneous GABAergic release still persisted and that a voltage-dependent Cl conductance contributed to burst termination. To test the hypothesis that epileptiform activity induced by lowering extracellular Ca$ sp{2+}$ was in part due to an alteration in the delicate balance between intracellular messengers, I investigated the effects of Ca$ sp{2+}$ (a) on the activity of hippocampal neurones, and (b) on the second messengers activated by acetylcholine (ACh), a neuromodulator known for its seizure promoting effects. Inhibiting cyclic nucleotide-dependent kinase activity was H-8 did not have any effect either on excitability of CA1 neurones or the excitatory actions of ACh. Activation of protein kinase C (PKC), a Ca$ sp{2+}$-binding protein, by phorbol esters enhanced high threshold Ca$ sp{2+}$ currents and subsequently facilitated neurotransmitter release (both excitatory and inhibitory) without significantly affecting ACh-induced effects. Inhibition of PKC by several dual PKC and Ca$ sp{2+}$/calmodulin-dependent kinase antagonists, such as H-7, sphingosine, and trifluoroperazine reduced inhibitory transmission and prevented ACh-induced effects. / The evidence presented in this thesis was combined with that in the literature to propose a model accounting for the seizure-promoting effects of ACh: that gangliosides and/or sphingolipids are metabolized to sphingosine upon muscarinic receptor activation leading to inhibition of both PKC and Ca$ sp{2+}$/calmodulin-dependent kinases.

This study investigates the role of substance P and NK-1 receptors in an animal model of neuropathic pain. Unlike naive animals, innocuous peripheral stimulation of neuropathic animals was determined to cause heterosegmental inhibition in the tail-flick test as well as increased plasma extravasation in the paw. These effects were prevented by administration of CP-96,345 before stimulation. Additionally, CP-96,345 or an antisense oligonucleotide against NK-1 receptors significantly alleviated mechanical allodynia in neuropathic animals. Finally, mass spectrum of lumbar spinal cord of neuropathic but not naIve animals showed a significant upregulation of substance P. We conclude that innocuous stimulation of a neuropathic area could trigger activation of NK-1 receptors, presumably due to binding of substance P. Furthermore, this activation of NK-1 receptors could be central to the perception of mechanical allodynia in neuropathic pain. These results justify the investigation of inhibiting the interaction between substance P and the NK-1 receptor for the treatment of drug resistant neuropathies.

ASP acts as a paracrine and autocrine signal to increase triglyceride synthesis in adipocytes. ASP administration results in more rapid postprandial lipid clearance. This present work consists of a series of experiments on complement C3 knockout mice (therefore ASP deficient mice) in vivo. The aim of this work is to understand the role of Acylation stimulating protein (ASP) in energy regulation. / Compared to wild type mice, ASP deficient mice have delayed triglyceride and fatty acid clearance, decreased fat mass and body weight with concurrent increases in food intake. Both male and female ASP deficient mice have increased oxygen consumption, an indication of increased energy expenditure. This effect was not dependent on the presence of leptin. On the other hand, the mechanism of increased energy expenditure was different in male and female ASP deficient mice. Female ASP deficient mice have increased movement while the male ASP deficient mice were found to have increased uncoupling protein-3 expression in muscle. Fat load tests in the male mice demonstrate ASP deficiency redirects absorbed energy from storage in adipose tissues towards utilization in liver and muscle. Acute administration of ASP normalizes this process. / The results from these studies suggest that ASP is a key hormone regulating lipid storage in adipocytes. Deficiency of ASP leads to energy repartition, decreased energy storage and increased energy expenditure. In short, ASP deficiency results in obesity resistance.

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.)