No description available.

No description available.

The proximal tubule of the renal cortical nephron is highly susceptible to intoxication by chemical agents. An in vitro system was developed to study directly the effects of nephrotoxic chemicals on this renal sub-organ fraction without the complication of extrarenal factors. Segments of proximal tubules were isolated by a mechanical method from the kidneys of young rabbits. Tubules obtained by this method retained biochemical, functional, and morphological features comparable to those existing in vivo. Preliminary acute susceptibility studies demonstrated that the isolated proximal tubule segments were sensitive to a variety of known nephrotoxic agents that target the proximal tubule. These agents include halogenated hydrocarbons, heavy metals, and a halogenated vinyl cysteine conjugate. Incubation conditions were optimized to maintain the viability of proximal tubule suspensions for up to four hours. Longer incubation times made it possible to establish a chronology of early tubule responses to chemical intoxication. Long term incubation of proximal tubule suspensions with two model nephrotoxins, cadmium chloride and S-(trans-1,2-dichlorovinyl)-L-cysteine, produced in vitro tubule response patterns similar to those reported in vivo for these agents. While not entirely representative of in vivo exposure conditions, suspensions of isolated proximal tubules are an easily obtained system that proved equally applicable as a screening technique for nephrotoxic compounds or as an in vitro system for delineating proximal tubule response to chemical insult.

No description available.

Intracellular microelectrodes have been used in conjunction with ion substitution, and agonists and inhibitors of known transport processes to investigate the mechanisms whereby ions cross the basal and apical cell membranes of the Malpighian tubules of Locusta. Values for basal, apical and transepithelial potentials in 'Normal' saline were -71.6 ± 0.3 mV, -82.6 ± 0.8 mV and +5.7 ± 1.0 mV (lumen positive) respectively. Ion substitution experiments, involving Na(^+),K(^+) and C1(^-) in the bathing media, indicated that the basal membrane was more permeable to K(^+) than Na(^+) and C1(^-). Two different electrical responses to high [K(^+)](_o) saline (the Type A and Type B response) were noted and these probably reflect distinct physiological states of basal membrane permeability. Experiments with ouabain and vanadate suggested that whilst Na(^+)+K(^+) ATPase activity, which has been demonstrated in microsomal preparations, was not significantly electrogenic, asymmetric ionic distribution across the basal membrane was partly maintained by thisenzyme Furthermore, 3-H ouabain-binding studies indicated that Na(^+)+K(^+) exchange 'pump' turnover was adequate to account for substantial entry and Na^ exit across the basal membrane. The electrochemicalgradient across the apical membrane suggests that exit from the cell must involve an active process with CI following passively. Data from ion substitution experiments and treatment with furosemide and bumetanide suggest that CI entry across the basalmembrane may be via cotransport with Na^ and/or K^. However, the+ —differential electrical responses to Na(^+) free and C1(^-) free salines question the role of Na(^+) in this process. The effects of c AMP, Ca(^2+) substitution and various inhibitors on basal and apical membrane potentials, taken in conjunction with the results referred to above, are discussed and a hypothetical model proposed whereby changes in intracellular Ca(^2+) and c AMP effect control of ion movements across the two cell surfaces.

No description available.

The convoluted (CPT) and straight (SPT) portions of the renal proximal tubule are susceptible to injury by a wide variety of chemical agents. These agents often affect the CPT or SPT selectively by proposed mechanisms usually attributed to tubular concentration, blood flow delivery patterns and tubuloglomerular feedback responses within the intact kidney. The innate cellular responses to chemical exposures remain virtually unexplored. Hence, the basic goal of this research was to develop an in vitro system that was conducive to examining the innate cellular differences in susceptibility between the CPT and SPT following in vitro exposure to mercuric chloride (HgCl₂), potassium dichromate (K₂Cr₂O₇)$ or hypoxic conditions. A renal cortical slicing technique was developed for these studies to position the CPT and SPT within discrete regions of slices made perpendicular to the cortical-papillary axis. An incubation vessel that could maintain the morophological and biochemical viability of slices for at least 12 hr was also developed. The selective necrosis of CPT induced by K₂Cr₂O₇ or hypoxic exposure, and SPT induced by HgCl₂, observed in vivo was reproduced in renal cortical slices exposed in vitro. Innate cellular uptake mechanisms were then investigated since the tissue distribution of each metal was found to be most concentrated within their respective injured cell type. The transport of PAH, TEA, phosphate, sulfate, glutathione and cysteine were examined as potential mechanisms for selective accumulation of these metals. K₂Cr₂O₇ caused a dose-dependent reduction in the uptake rate of sulfate by cortical slices, while phosphate, PAH, and TEA uptake were unaffected. Although HgCl₂ has a high affinity for sulfhydryl groups its uptake as a complex to glutathione or cysteine was not enhanced. HgCl₂ also had no affect on the uptake rate of PAH or TEA even though both HgCl₂ and K₂Cr₂O₇ were able to reduce the steady state accumulation of these organic substrates.

Membranous tubules are very abundant structures in living cells and form or are part of most intracellular organelles. The Golgi apparatus is mainly formed by tubules, which adopt different geometries and conformations. However, their physiological role has not yet been established and this is mainly due to the almost absolute lack of knowledge about the biochemical mechanisms regulating their formation, maintenance and disruption. The aim of this thesis was to investigate in a systematic way these mechanisms. The first step has been to set up an in vitro morphological assay suitable for the visualisation of Golgi-associated tubules in isolated Golgi stacks. This assay was based on electron microscopy and specifically on negative staining of whole-mount preparations. It allowed both qualitative and quantitative analysis of the morphological changes of Golgiassociated tubules after in vitro incubations. This assay was then used for screening several molecules or experimental conditions for their effect on tubular homeostasis. Among them, the most significant was BARS (BFA-dependent ADP-Ribosylation Substrate), a protein previously implicated in the maintenance of Golgi architecture. BARS has been found to cause the selective breakdown of the tubular part of the Golgi complex promoting fission events which convert the tubular structures into clusters of vesicles. This effect correlated with the enzymatic activity of BARS, which acts as an acyl-CoA dependent lysophosphatidic acid acyl transferase (LPAAT), increasing phosphatidic acid (PA) levels in Golgi membranes. This suggests that local modifications of the composition of the lipid bilayer is a possible mechanism for the fission of membranous tubules.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.