Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

Uptake of bumetanide into rat liver cells was investigated using isolated hepatocytes and primary cell cultures. The kinetics of [3H]-bumetanide uptake revealed two saturable components in addition to an unsaturable component. Saturable bumetanide uptake consists of a high-affinity, sodium-dependent uptake and a low-affinity transport system. Bumetanide uptake into isolated rat hepatocytes is energy dependent and temperature sensitive. At low temperatures, bumetanide uptake is due to diffusion with a permeability coefficient of 1.16 x 10(-6) cm/s. In primary liver cell cultures, uptake of bumetanide decreases rapidly over 3 days. AS-30D ascites hepatoma cells do not take up bumetanide but bind small amounts of the loop diuretic. Hepatocytes metabolized bumetanide extensively. The metabolites were secreted into the surrounding incubation buffer. Two hydroxylated and at least one conjugated biotransformation product could be separated by thin-layer chromatography. Isolated rat hepatocytes possess carrier proteins for uptake of bumetanide and very likely also for uptake of other loop diuretics like furosemide, piretanide, and torasemide. Several inhibitors of multispecific transport systems in the kidney and liver were tested as potential inhibitors of hepatocellular bumetanide or furosemide uptake. Probenecid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, iodipamide, digitoxin, bile acids, and bromosulfophthalein inhibited uptake of loop diuretics. Inhibition by taurocholic acid was competitive with a Ki of 24 microM. Taurocholic acid inhibited [3H]bumetanide uptake in the presence but not in the absence of Na+. Deoxycholic acid and bromosulfophthalein were noncompetitive inhibitors of hepatocellular bumetanide uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Over the years, immortalized rodent β-cell lines such as RIN, HIT, MIN, βTC, and INS-1 have been used to investigate pancreatic β-cell physiology using conventional two-dimensional (2D) culture techniques. However, physical and physiological limitations inherent to 2D cell culture necessitates confirmatory follow up studies using sentient animals. Three-dimensional (3D) culture models are gaining popularity for their recapitulation of key features of in vivo organ physiology, and thus could pose as potential surrogates for animal experiments. In this study, we aimed to develop and characterize a rat insulinoma INS-1 3D spheroid model to compare with 2D monolayers of the same cell line. Ultrastructural verification was done by transmission electron microscopy and toluidine blue staining, which showed that both 2D monolayers and 3D spheroids contained highly granulated cells with ultrastructural features synonymous with mature pancreatic β-cells, with increased prominence of these features observed in 3D spheroids. Viability, as assessed by cellular ATP quantification, size profiling and glucose utilization, showed that our spheroids remained viable for the experimental period of 30 days, compared to the limiting 5-day passage period of INS-1 monolayers. In fact, increasing ATP content together with spheroid size was observed over time, without adverse changes in glucose utilization. Additionally, β-cell function, assessed by determining insulin and amylin secretion, showed that the 3D spheroids retained glucose sensing and insulin secretory capability, that was more acute when compared to 2D monolayer cultures. Thus, we were able to successfully demonstrate that our in vitro INS-1 β-cell 3D spheroid model exhibits in vivo tissue-like structural features with extended viability and lifespan. This offers enhanced predictive capacity of the model in the study of metabolic disease, β-cell pathophysiology and the potential treatment thereof.

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