Using epifluorescence microscopy I have characterized the transport of the fluorescent organic cation NBD-TMA⁺, [2-(4-nitro-2,1,3-benzoxadiaxol-7-yl)aminoethyl]trimethylammonium. NBD-TMA⁺ has structural characteristics common to other secreted organic cations and it is fluorescent (λₑₓ = 458 nm; λ(em) = 530 nm). Transport of NBD-TMA⁺ by rabbit renal proximal tubules was time dependent, saturable, and inhibited by TEA⁺, cimetidine, and procainamide. These results provide strong evidence that the fluorescent organic cation, NBD-TMA⁺, is transported by the classical organic cation transporter and can be used to monitor organic cation transport in renal proximal tubules or cells expressing organic cation transporters. Additionally, a stable line of HeLa cells expressing hOCT1 was used to investigate the structural and chemical characteristics influencing substrate binding to the transporter. I found that the systematic rotation of a planar hydrophobic mass about a vertical axis aligned with the positively charged nitrogen reduced the binding affinity of substrate for hOCT1 suggesting that hOCT1 possesses a lateral steric limitation not seen in the apical organic cation transporter. Furthermore, the addition of supraplanar hydrophobic mass to a planar chemical structure was found to increase the binding affinity for a substrate substantially more that expected for the increased hydrophobicity suggesting that the binding site for hOCT1 is not simply planar in nature, but may be better described as a pocket.

This thesis addresses the role of diaphragmatic afferents in the regulation of breathing. The activation of diaphragmatic thin-fiber afferents by bradykinin, a physiological substance produced during muscle contraction, caused a stimulation and a redistribution of inspiratory motor drive, and a decrease in inspiratory time. These afferents are also involved in the regulation of airway smooth muscle tone, since tracheal tension and lung resistance decreased during electrical activation of the phrenic nerve. To mimic in vivo activation of mechanoreceptive afferents, phrenic nerve stimulation was performed during inspiration or expiration. Similar increases in inspiratory motor drive and respiratory timing occurred during inspiratory and expiratory phrenic stimulation. In addition, vagal input potentiated the stimulatory effect of inspiratory phrenic, but not tibial, nerve activation. Prolonged activation of diaphragmatic thin-fiber afferents by ischemia caused inspiratory motor drive to increase and then decrease to baseline values. These findings indicate either a depletion of neurotransmitter substance within afferent pathways, or the development of central inhibition of ventilatory drive. In summary, diaphragmatic thin-fiber afferent activity is an important modulating influence in the control of breathing.

The delayed rectifier potassium current IKr (hERG) participates in repolarization of the cardiac action potential and is implicated in one form of inherited Long QT Syndrome. The goal of this study was to characterize the biophysical properties of IKr in isolated rabbit ventricular myocytes and hERG expressed in a mammalian cell line using mathematical, electrophysiological and molecular biological techniques. Incorporation of results into a mathematical gating model of IKr revealed that the current did not follow the typical Hodgkin-Huxley type gating formulation and therefore comprised a gating model paradox. Furthermore, it was found that different groups of divalent cations selectively affected specific kinetic properties of I Kr, with the relief of rectification of the current by the Cd 2+-like class of divalent cations (Cd2+, Ni 2+, Co2+ and Mn2+; mM range) and the block of the current by Zn2+ (0.1--1 mM) being the most prominent effects. The biophysical properties of hERG were found to be more sensitive to Cd2+ compared to rabbit IKr (muM as opposed to mM concentrations). The relief of rectification of native rabbit IKr and expressed human hERG by the Cd2+-like group of divalent cations and block of rabbit IKr by Zn2+ may be attributed to specific interactions with distinct sites associated with different regions of the channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane chloride channel expressed in epithelial tissues. While protein kinase A (PKA) is the main activator of CFTR, patch clamp data have suggested a permissive role for PKC in modulating PKA responses. To clarify the role of PKC in CFTR regulation we used PCR mutagenesis to construct a CFTR mutant (9CA) in which the serines or threonines in nine consensus sequences for PKC phosphorylation were replaced by alanines. Its expression in stably transfected baby hamster kidney (BHK) cells was consistently lower than that of wild-type (WT) CFTR. Nevertheless, 9CA cells displayed a robust cAMP-stimulated iodide efflux which was comparable in magnitude to that of WT CFTR cells when normalised for protein expression level. The cpt-cAMP concentration-response relationship in intact cells was similar for WT and 9CA CFTR (EC50 ≈ 100muM), however the onset of iodide efflux from cells expressing the mutant was markedly delayed at all cpt-cAMP concentrations tested. This delay was mimicked by pre-treating WT cells with the PKC inhibitor chelerythrine (10muM), and chelerythrine did not cause further slowing of iodide efflux from 9CA cells. In vitro phosphorylation by PKA and PKC was similar for WT and 9CA CFTR and phosphorylation in the presence of both kinases was additive. / A major impediment to understanding CFTR structure/function has been the difficulty of obtaining sufficient purified CFTR protein for biochemical studies. In an attempt to overcome this problem we expressed a His-tagged CFTR construct in the methylotrophic yeast Pichia pastoris. The final yield of CFTRHis10 following solubilization of Pichia membranes in 0.5% lysophosphatidylglycerol (LPG) and nickel chelate chromatography was ∼20mug/litre. CFTRHis10 was not glycosylated in this yeast system. Both PKA and PKC phosphorylated semi-purified CFTRHis10 in vitro and phosphorylation in the presence of both kinases was additive.

Leukotriene B$ sb4$ (LTB$ sb4)$ has previously been reported to be metabolized by porcine polymorphonuclear leukocytes (PMNL) to 10,11-dihydro-LTB$ sb4$ and 10,11-dihydro-12-oxo-LTB$ sb4$ (Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369). The 10,11-dihydro-LTB$ sb4$ fraction, prepared as described above, was resolved into two components by normal-phase (NP) high pressure liquid chromatography (HPLC). These products were identified as 10,11-dihydro-LTB$ sb4$ and 10,11-dihydro-12-epi-LTB$ sb4$ by gas-chromatography mass-spectrometry (GC-MS) and by co-chromatography with authentic chemically synthesized compounds. In the presence of NAD$ sp+,$ a microsomal 12-hydroxyeicosanoid dehydrogenase converted LTB$ sb4$ to 12-oxo-LTB$ sb4,$ which was identified by GC-MS. This enzyme catalyzes the initial rate-limiting step in the formation of dihydro metabolites of LTB$ sb4.$ 12-Oxo-LTB$ sb4$ was rapidly metabolized to 10,11-dihydro-12-oxo-LTB$ sb4$ by a cytosolic 10,11-reductase in the presence of NADH or NADPH. LTB$ sb4$ was not converted to any products by this fraction. / 12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) was metabolized by intact porcine PMNL to 12-oxo-5,8,14-eicosatrienoic acid, 12(R)-hydroxy-5,8,14-eicosatrienoic acid and 12(S)-hydroxy-5,8,14-eicosatrienoic acid, which were identified by GC-MS and nuclear magnetic resonance spectroscopy. The latter two compounds were separated by NP-HPLC following derivatization with methoxy-(trifluoromethyl)phenylacetic acid and were identified by co-chromatography with authentic compounds. 12(S)-HETE was metabolized by 12-hydroxyeicosanoid dehydrogenase, in the presence of NAD$ sp+,$ to 12-oxo-ETE, identified by its chromatographic and UV spectral properties. 13-Hydroxy-9,11-octadecadienoic acid (13-HODE) was metabolized by porcine PMNL to 13-hydroxy-9-octadecenoic acid and 13-oxo-9-octadecenoic acid which were identified by GC-MS. Specificity studies indicated that the porcine PMNL 12-hydroxyeicosanoid dehydrogenase preferentially oxidizes substrates with a 12-hydroxyl group preceded by at least two conjugated double bonds and followed by a 2-cis-octenyl group.

This thesis focuses on the renin-angiotensin system and atrial natriuretic factors (ANF) two hormonal systems which are stimulated and which may exert important antagonizing actions on the regulation of mean arterial pressure (MAP) and body fluid homeostasis during pregnancy. All experiments were conducted in healthy nonpregnant and/or pregnant (gestational age of 105 to 140 days, term = 145 days) mixed breed ewes, using a facility designed for studies of large animals. Findings from the first study with unilateral denervated kidneys provide evidence that renal nerves are a necessary component in the control of renin secretion in both nonpregnant and pregnant ewes. The second study demonstrates that angiotensin II (AngII) and ANF do not account for the dramatic suppression of renin secretion in response to the reduction of renal perfusion pressure in sheep with bilateral renal denervation. The data from these two studies suggest that the renal baroreceptors influence renin secretion indirectly through activation of renal afferents rather than by a direct action on the juxtaglomerular apparatus. In order to study the possible effects of increased plasma AngII on ANF production during pregnancy, four doses of AngII (0.5, 5, 20, 40 ng/kg/min) and nitroprusside were simultaneously infused to separate indirect hemodynamic actions on ANF secretion from direct hormonally mediated effects on ANF secretion by AngII. The data clearly show that AngII increases plasma ANF in a dose-dependent manner but: only in the presence of the AngII pressor effect. A striking finding was the demonstration that the natriuretic and diuretic responses to intrarenal artery infusion of three doses of ANF (0.3, 1.5, 3.0 pmol/kg/min) are increased during ovine pregnancy and these responses are solely limited to actions on the distal part of the nephron without altering renal vascular or glomerular function. Finally, a study with 10 days of intrarenal artery infusion of low dose AngII (1 ng/kg/m

This thesis examines changes in the mechanical behaviour of the canine and human respiratory systems to changes in lung volume below normal functional residual capacity (FRC). In open chested dogs lung elastance (E$ sb{ rm L}$) increased and lung resistance (R$ sb{ rm L}$) changed little with decreases in positive end-expiratory pressure (PEEP) of the ventilatory circuit. The dominance of plastoelastic lung tissue properties at low lung volumes was used to interpret the lack of change in R$ sb{ rm L}$. Computed tomography demonstrated that pleural effusion (PE) created atelectasis in dependent caudal lung regions which contributed to the overall lung volume loss. PE produced a decrease in only lung vertical height while chest wall dimensions changed both vertically and horizontally. E$ sb{ rm L}$ and R$ sb{ rm L}$ increased while elastance and resistance of the chest wall were little affected by these shape and density changes. In close-chested, anesthetised, paralysed, ventilated humans a decrease in PEEP below normal FRC caused an increase in R$ sb{ rm L}$, E$ sb{ rm L}$ and both chest wall elastance and resistance. Median sternotomy caused E$ sb{ rm L}$ to increase with increasing PEEP while the negative volume dependence of R$ sb{ rm L}$ remained. Most of the difference between open-chested and closed-chested E$ sb{ rm L}$ was presumably due to lung collapse in the open-chested state.

A genetic polymorphism of sheep red cells characterized by differences in the intracellular K$ sp+$ concentration of their mature red cells reflects differences in their Na/K pump activity as well as pump kinetics. This polymorphism is linked to the ML blood group system, as follows. Homozygous high-K$ sp+$ (HK) cells possess only M antigen, whereas homozygous low-K$ sp+$ (LK) cells have an antigen, L$ rm sb{p},$ which is an endogenous inhibitor of the Na/K pump since isoimmune antiserum, anti-L$ rm sb{p},$ stimulates the Na/K pump of LK cells by relieving this inhibition. In this study, it is shown that the $ rm L sb{p}$ antigen is a molecular entity distinct from the pump. Thus, anti-$ rm L sb{p}$ stimulated the activity (ouabain-sensitive $ rm sp{86}Rb sp+(K sp+)$ influx) of exogenous rat kidney pumps incorporated by fusion of kidney microsomes into immature LK red cells. Moreover, the $ rm L sb{p}$ antigen is responsible for the changes in pump kinetics associated with maturation of LK sheep red cells. The evidence is that treatment of immature LK cells with either anti-L$ rm sb{p}$ or trypsin (which cleaves the $ rm L sb{p}$ antigen) prevents the maturation-associated change in kinetics of the pumps, normally from one of no inhibition (immature cells) to that of marked inhibition by intracellular K$ sp+$ (mature cells). In addition, it was shown that kidney pumps delivered into immature LK red cells develop sensitivity to inhibition by intracellular K$ sp+$ following maturation of the cells in vitro. Mature LK cells have fewer pumps than mature HK cells and, therefore, another question addressed was whether the $ rm L sb{p}$ antigen promotes the greater loss of pumps in LK red cells during maturation. It was observed that modification of the $ rm L sb{p}$ antigen of immature LK cells with either anti-L$ rm sb{p}$ or trypsin diminished the subsequent loss of pumps during maturation in vitro. It was shown also that there is little decline in the number of $ rm

Although the gonadotropins are necessary for follicular development, the spatio-temporal specific differentiation of the constituent granulosa cells and the process of selection for ovulation may be regulated by other intrafollicular mechanisms. Gap junction-mediated intercellular communication between granulosa cells has been previously reported and may play a role in spatio-temporal differentiation and/or follicular selection. In order to elucidate the function of intercellular communication in follicular development, granulosa cell cultures were treated with agents known to disrupt gap junctions (uncouplers): the retinoids and the alkanols. Retinoic acid, heptanol annd octanol inhibited the expression of luteinizing hormone (LH) receptors and the accumulation of progesterone in the culture. The expression of LH receptors and the production of progesterone are crucial follicular functions. Thus, the inhibition of these functions by uncouplers provides the first evidence that the establishment of gap junctional communication may be an integral component in follicular development.

No description available.