The insulin resistance syndrome is a multifaceted condition characterized by a clustering of metabolic and cardiovascular abnormalities, including insulin resistance of skeletal muscle glucose metabolism, hyperinsulinemia, glucose intolerance, dyslipidemia, essential hypertension, and central adiposity. Individual interventions with antioxidants or endurance exercise training enhanced insulin action on skeletal muscle and whole body insulin sensitivity in the markedly insulin-resistant, hyperinsulinemic, and dyslipidemic obese Zucker (fa/fa) rat. Individually, antioxidant treatment and exercise training by the obese Zucker rat resulted in a decrease in protein carbonyls (reflective of local oxidative stress), plasma free fatty acids, and intramuscular triglycerides, as well as an upregulation of the protein expression of insulin receptor substrate-1 (IRS-1), a critical component of the insulin signaling pathway. Whereas exercise training alone enhanced the protein expression of GLUT-4 glucose transporter isoform, this protein expression was not affected by antioxidant treatment. Most importantly, this study has investigated the interactions of antioxidant and exercise training in combination on whole body insulin sensitivity and skeletal muscle insulin action in the obese Zucker rat. The combination of antioxidant and exercise training functioned in an additive fashion and brought about the greatest increases in insulin action on skeletal muscle glucose transport activity and IRS-1 protein expression compared with either intervention individually. In addition, the IRS-1 protein expression, following the individual or combined intervention of antioxidant and exercise training, was correlated with insulin-mediated glucose transport in skeletal muscle. It is therefore likely that, in response to insulin, the downstream signaling from the expanded IRS-1 protein pool, in skeletal muscle from obese Zucker rats treated with antioxidant and exercise training in combination, acts on the expanded GLUT-4 pool (derived from exercise training) to bring about the greatest incorporation of GLUT-4 into the plasma membrane, with a corresponding enhancement of glucose transport activity. This study supports the utility of the combination of exercise training and antioxidant for the prevention and treatment of insulin resistance and type 2 diabetes.

Arcades are blood vessels that form direct connections between two arteries or arterioles. Because they are supplied with blood from two sources, arcades can function as alternative flow pathways following obstruction of arteries or arterioles, as in coronary and peripheral vascular disease and stroke. In response to changes in blood flow or metabolic conditions, vascular networks undergo structural adaptation or remodeling, which includes structural changes of the existing vessels and growth of new vessels. Following obstruction of a blood supply, arcade vessels may adjust their internal diameters chronically to convert the alternative pathways into main blood distribution vessels. The overall goal of this dissertation was to examine structural changes in the internal diameter of a single arcade artery and the arterioles of an arcade network following changes in blood flow, using experimental and theoretical approaches. Diameter changes of the mouse gracilis arcade artery were observed up to 56 days following resection of one of its two blood supplies. Overall, diameters increased to a maximum around day 21 and then declined. The diameter changes were spatially non-uniform, being largest towards the point of resection, providing transiently increased perfusion to the most affected regions. Observed diameter changes were compared with predictions of a theoretical model, in which diameter varies in response to stimuli derived from local metabolic and hemodynamic conditions. Good agreement was found when effects of a time-delayed growth stimulus in regions of reduced perfusion were included, with a delay of about 7 days. The effectiveness of arcades in maintaining perfusion both immediately following obstruction and after structural adaptation in the arteriolar arcade network between two feed artery branches of the pig triceps brachii muscle was examined. Morphometric data from vascular casting and published data were used to develop a computational model for the hemodynamics and structural adaptation of the network in response to local stimuli. The results show that the arcades provide alternative flow pathways to the region initially supplied by the obstructed branch and that structural adaptation can lead to improved flow restoration following interruption of blood flow.

Potassium channels govern the duration and frequency of excitable membrane events, and thus may regulate voltage-dependent signals that are important in neuronal development. This study assesses the developmental expression of two classes of K⁺ channels in vivo and in vitro in the rat cerebellum. In vivo, the level of mslo-related transcript for the Ca²⁺-dependent K⁺ channel (KCa) was shown by Northern analysis to be upregulated during development, whereas transcripts for delayed rectifier (KD) channels remained fairly constant. The same pattern of in vivo development was demonstrated with functional assays by expression in Xenopus oocytes of poly A-enriched RNA isolated from postnatal rat cerebella. In vitro, single channel studies of Purkinje neurons showed that KCa channel activity was increased during development and KD channel activity remained stable. Although the semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that the level of transcripts of the KCa channel sequence remained constant in control culture, the developmental pattern that was seen in vivo was mimicked in vitro when cultures were treated chronically with tetraethylammonium (TEA, 1mM). Chronic treatment with 10 mM extracellular KCl resulted in an upregulation of KCa transcripts similar to that seen with chronic TEA. The stimulatory effects of TEA or KCl were negated in low external calcium (0.1 mM), suggesting that KCa transcript levels were influenced by depolarization and calcium entry. The KCa channels may in part contribute to the mature electrical properties of Purkinje neurons. This was supported by evidence that developmental trends in cellular firing activity were antagonized by decreased KCa channel abundance caused by chronic treatment with TEA. Voltage-gated Ca²⁺ channels (N, R and P type) were developmentally down-regulated at the transcriptional level in control cultures. Chronic treatment with TEA increased the transcript levels for N and R type Ca²⁺ channels, but not for P type, suggesting that the various types of Ca²⁺ channels were differentially regulated. Ca²⁺ signaling plays a key role in neuronal development in many cells. The KCa and Ca²⁺ channels regulate Ca²⁺-entry, and may thus influence the neuronal differentiation.

The mammalian upper airway includes the larynx, pharynx, and nose. Respiratory-related contraction of the skeletal muscles situated in and around these regions influences upper airway diameter and compliance. The pharynx is the most collapsible upper airway segment, and its diameter and compliance depend in part on tongue position and stiffness. These parameters are controlled by the genioglossus (GG) muscle, which protrudes the tongue, and the hyoglossus (HG) and styloglossus (SG) muscles, which retract the tongue. Prior work has focused almost exclusively on the GG, leaving a gap in the literature regarding the respiratory control and function of the tongue retractors. Accordingly, our overall purpose was to test the hypothesis that the tongue protrudor and retractor muscles are co-activated during inspiration and that co-activation promotes airway patency. Experiments were conducted using supine, anesthetized, tracheotomized rats. Tongue movements were quantified as either protrusive or retractive by connecting the tip of the tongue to a force transducer. The protrudor and retractor muscles were co-activated during quiet breathing and their activities increased in parallel when breathing was stimulated with high CO2 or low O 2. Co-activation of protrudor and retractor muscles was always accompanied by tongue retraction. Neural drive to both GG and HG muscles was increased in parallel when lung volume feedback was removed by single breath tracheal occlusion. The functional significance of tongue muscle co-activation was examined using an isolated upper airway preparation. Co-activation increased airflow rates and stiffened the airway, whereas selective protrudor muscle activation increased airflow but did not alter airway stiffness. A standard fatigue protocol was used to examine the influence of hypoxia on the endurance performance of tongue protrudor and retractor muscles; the results indicate that hypoxia attenuates tongue muscle endurance, possibly via impaired neuromuscular transmission. It is concluded that, in the rat, (1) the tongue protrudor and retractor muscles are co-activated during inspiration, and respond in parallel to increases in respiratory drive; (2) tongue muscle co-activation results in tongue retraction and stiffening of the pharyngeal airway, and (3) the endurance of the tongue muscles is impaired during hypoxia.

The goal of this dissertation was to test the hypothesis that fibroblast growth factor-2 (FGF2) is required during diseased-related vascular growth and remodeling in the adult organism. Given previous research, it is generally assumed that FGF2 is an important regulator of vessel growth during various pathophysiological processes (e.g. tissue ischemia, vessel injury, and flow-dependent remodeling). However, such studies only indirectly implicate FGF2 in vascular adaptation and remodeling. In contrast, experiments using mice with a targeted disruption of the Fgf2 gene have allowed direct determination of the biological roles of endogenous FGF2. Thus, experimental models of flow-dependent remodeling and ischemic revascularization were used to compare the responses of Fgf2⁻/⁻ and Fgf2⁺/⁺ mice to directly identify the function of FGF2 during vascular adaptation in the adult animal. Surprisingly, the lack of FGF2 did not appear to affect vascular growth in these models. First, using a novel model of flow-dependent remodeling, Fgf2⁻/⁻ mice had equivalent carotid artery adaptation in response to both high-flow and low-flow was as wildtype counterparts. Second, angiogenesis and arteriogenesis were not different between the ischemic limbs Fgf2⁺/⁺ and Fgf2⁻/⁻ mice, demonstrating that FGF2 is not required for vascular adaptation in response to ischemia. However, these experiments led to the observation that reactive hyperemia was impaired in ischemic limb of Fgf2⁻/⁻ mice. These results indicate that vessel responsiveness is altered in the collateral circulation of the ischemic Fgf2⁻/⁻ limb. This possible identification of FGF2 as a "functional" factor in the collateral circulation suggests a novel, non-mitogenic role for endogenous growth factors. Finally, Fgf2⁻/⁻ mice had altered gene expression in the ischemic limb as evaluated using cDNA microarrays. The significance of differential gene expression in the absence of FGF2 is unknown. It is unclear whether such changes in gene expression are related to the FGF2 hyperemia phenotype or whether they are related to an unknown phenotype present in the ischemic limb of Fgf2⁻/⁻ mice. Overall, this dissertation provides new evidence that endogenous FGF2 has important actions in the remodeling vasculature during ischemic revascularization. Specifically, endogenous FGF2 appears to modulate vascular reactivity of the collateral circulation of the hindlimb.

Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and an endoderm-derived signal acting on mesoderm. In this study, experiments were designed to investigate the potential role of TGFβ superfamily members in regulating these early steps of heart muscle cell development. While activin or TGFβ can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2/BMP-4 can induce cardiac myocyte formation in mesoderm in a variety of contexts, but show no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFβ and BMP-2/BMP-4 therefore have distinct and reciprocal inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and the anterior lateral endoderm, respectively. Experiments with follistatin and noggin provide additional evidence that BMP signaling lies downstream of activin signaling in the cardiac myogenesis pathway. BMP-2 or BMP-4 inhibit cardiac myogenesis prior to stage 3, demonstrating a dual role for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFβ signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, act on emerging mesoderm within cardiogenic regions. These signals, mediated in part by BMPs, activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation. Members of the FGF and the EGF-related CFC families may also participate in this pathway. FGF-2/FGF-4 can induce posterior region epiblast to form heart muscle cells, and the CFC family member Cripto can convert posterior lateral mesoderm to a myocardial phenotype. The role of these factors in the cardiac myogenesis pathway is unclear.

A newly developed epifluorescense microscopy system has been employed to measure net transepithelial secretion of fluorescein (FL) in real time in isolated perfused S2 segments of rabbit renal proximal tubules. Net FL secretion (K(t), ∼4 μM, and J(max), ∼280 fmol·min⁻¹·mm⁻¹) shares the same transport system with that of para -aminohippurate (PAH). The basolateral Na-DC cotransporter supports ∼25% of the "basal" FL secretion in the absence of exogenous αKG via recycling of αKG that has been exchanged for FL. Physiological αKG concentrations in the bath (∼10 μM) or in the perfusate (∼50 μM) stimulated net secretion of FL by ∼30 or ∼20%, respectively. These data indicate that the basolateral Na-DC cotransporter supports ∼42% of the net FL secretion. The luminal and basolateral effects of physiological concentrations of αKG were additive. Together, the basolateral and luminal Na-DC cotransporters can directly support ∼50% of the net FL secretion, apparently, by their establishing and maintaining the outwardly directed αKG gradient responsible for driving basolateral FL/αKG exchange. The remaining ∼50% would be maintained by metabolic production of αKG in the cells. Adding of 100 nM phorbol 12-myristate 13-acetate (PMA), a known PKC activator, to the bath decreased steady-state secretion of FL by ∼30% after 25 min incubation. This inhibition was irreversible and increased to ∼60% 25 min following removal of PMA. The inhibition produced by PMA was blocked when 100 nM of either staurosporine (ST) or bisindolylmaleimide I (BIM), both known PKC inhibitors, was added to the bath. ST or BIM alone had no significant effect on FL secretion, suggesting that the basal FL secretion was not under the influence of PKC. Adding of 1 μM of either the peptide hormone bradykinin (BK) or the α₁-receptor agonist phenylephrine (PE), to the bath both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, inhibited FL secretion by ∼22% and ∼27%, respectively. The inhibition was completely reversible after removal of BK or PE. In conclusion, PKC negatively regulates the net secretion of FL in rabbit renal proximal tubules. The data indicate that BK or catecholamines can play a physiological role in regulating organic anion secretion via PKC activation.

Female insects produce eggs that are nutrient-rich. For most Lepidoptera, protein is acquired during larval feeding. Insects use pre-ingestive, post-ingestive and developmental strategies to meet nutritional needs. In this dissertation I examine the importance of these strategies to females of two related lepidopteran species differing in their adult feeding. Adult Heliothis virescens (Family Noctuidae) ingests nectar. The sexes were not distinguished according to their selective feeding behavior, but females accumulated more protein and carbohydrate. When restricted to diets, females ate and accumulated more protein on high protein diets. There were no sexual differences on high carbohydrate diets. Results indicate that female H. virescens larvae accumulate protein by regulating both intake and post-ingestive processing on high protein foods. Adult Estigmene acrea (Family Arctiidae) do not feed. The sexes did not differ in their selective feeding behavior. When restricted to diets, female E. acrea increased ingestion of diets unbalanced in protein and carbohydrate leading to greater intake of both nutrients but only accumulated more protein. Lastly, E. acrea prolong juvenile development if too small at later stages effectively extending their period of ingestion and processing. As previously shown protein-derived growth in female H. virescens progressively increased as dietary protein levels increased. Storage protein, a component of protein-derived growth, was found to similarly increase but was more abundant in females across all pupal stages and diets. Pharate adults retained a portion of total storage protein with females retaining greater levels presumably toward egg provisioning. Growth of both H. virescens and E. acrea was highly clustered compared to ingestion values suggesting post-ingestive processing of nutrients to regulate growth. Males and females of both species efficiently utilized carbohydrate except at high ingestion. Females of both species utilized nitrogen more efficiently than did males at all ingestion levels, contributing to their greater protein accumulation. The manner of post-ingestive processing by these two species reflects differences in their larval diet. Clearly, my studies show that female caterpillars regulate both nutrient consumption and post-ingestive physiology to accumulate greater reserves.

The respiratory muscles play a role in respiratory failure when the efficient performance of the work of ventilation and/or their supply of metabolic substrates is disrupted. In this report a model of inspiratory muscle action is presented. The inflationary pressure applied to the lungs and the lung apposed rib cage is partitioned into two parts. One component is attributable to the action of rib cage muscles and the other is due to the interaction between upper and lower rib cage compartments. These contributions were found to be equal. / The role of afferent impulses travelling in the phrenic nerve in the control of respiratory muscle activity was investigated by electrical stimulation of its central cut end. Activation of these fibres exerts a non-uniform effect on the activities of the upper airway, rib cage and abdominal muscles and may influence respiratory muscle recruitment. / The roles of blood flow and oxygen delivery in determining diaphragm function was investigated. The rate at which diaphragmatic fatigue develops is diminished at high rates of blood flow and this effect is not related to the associated increase in oxygen delivery. The critical oxygen delivery at which oxygen consumption becomes supply dependent is the same for the resting diaphragm as for the rest of the body tissues. Activation of the diaphragm results in a higher critical oxygen delivery, however, this effect is mitigated by an increase in the critical oxygen extraction ratio. / The role of nitric oxide in regulating diaphragmatic blood flow and oxygen uptake was investigated by infusion of N$ sp{G}$-nitro-L-arginine. This treatment increased diaphragmatic vascular resistance, reduced the duration and magnitude of reactive vasodilation and increased the oxygen consumption and critical extraction ratio in the contracting diaphragm.

The catalytic $ alpha$ subunit of the heterodimeric Na,K-ATPase comprises three distinct isoforms which are expressed in a tissue-specific manner. For example, the $ alpha sb1$ isoform can be detected in virtually all mammalian tissues, whereas the appearance of the $ alpha sb2$ and $ alpha sb3$ isoforms is more restricted to particular tissues such as muscle and nervous tissue. Previous comparative functional studies of Na,K-ATPases isolated from various tissues indicated that there are differences, including apparent cation affinities, among these enzymes. While these differences often appear to correlate with the presence of distinct isozymes, their precise molecular bases remain to be determined. Moreover, certain studies suggest that the behavior of the same isoform can vary from tissue to tissue (e.g., the erythrocyte versus the kidney, both of which contain only the $ alpha sb1$ isoform). An hypothesis that may explain these observations is that the cell-specific membrane environment influences Na,K-ATPase activity. To investigate this possibility, polyethylene glycol-mediated membrane fusion was used to deliver pumps from high-specific-activity microsomes derived from various tissues into mammalian erythrocyte membranes. The success of this methodology was verified using two distinct experimental systems. In the first system, rabbit sarcoplasmic reticulum Ca-ATPase was delivered into human erythrocyte membranes. Cellular Ca$ sp{2+}$ uptake fueled by extracellular ATP was used as a measure of the functional delivery of the Ca-ATPase into these membranes. In the second system, ATP- and cardiac glycoside-dependent rubidium fluxes verified the functional delivery of axolemma or kidney Na,K-ATPases into mammalian erythrocytes. Among these studies was a series of experiments demonstrating that the L$ rm sb{p}$-antigen of sheep erythrocyte membranes is a distinct membrane component that interacts with and alters the behavior of rat kidney pumps fused into LK she