Pancreatic ß-cells are heterogeneous in their secretory responsiveness, glucose sensitivity and metabolic rate. A diminished and delayed first-phase insulin release is an early sign of failing ß-cells in diabetes. Mechanisms controlling functional characteristics, such as lag time for insulin release or magnitude of the response in each individual cell are unknown. To find out whether the heterogeneity represents a random phenomenon in ß-cell or is a manifestation of reproducible characteristics, we compared parameters of Ca2+ response in Fura-2 labelled ob/ob mouse ß-cells during two consecutive stimulations with glucose. Lag times, as well as peak heights and nadirs of initial lowering showed a strong correlation between the first and second stimulation. Thus, timing and magnitude of the early Ca2+ response were specific for each cell. ß-Cells from lean mice, diabetic db/db mice and rats also showed cell-specific responses characteristics. This indicates that a cell-specific Ca2+ response to glucose is common in rodent ß-cells, both normal and diabetic. Another question was whether aggregated ß-cells show cell-specific responses. Using the same protocol as for dispersed ß-cells, we analysed Ca2+ responses in clusters of different size and in intact islets from ob/ob and lean mice. Correlations were found between the first and second stimulation for timing and magnitude of [Ca2+]i rise, and for the initial lowering. Next, we tested if the ß-cell response is cell-specific, when induced at different steps of the stimulus-secretion coupling. The glycolytic intermediate glyceraldehyde, the mitochondrial substrate KIC, the KATP-channel blocker tolbutamide and arginine were used as tools. [Ca2+]i changes were studied in dispersed ß-cells from lean, ob/ob and db/db mice. NADH responses to glucose and KIC were analyzed as a measure of metabolic flux. The correlation between Ca2+ and insulin response from individual ß-cells was tested using Fluo-3 and Fluozin-3. Both timing and magnitude of calcium responses were cell-specific in lean mouse ß-cells with all tested secretagogues. ß-Cells from ob/ob and db/db mice showed cell-specific timing of Ca2+ responses to glyceraldehyde but not to KIC, tolbutamide or arginine. However, ob/ob mouse ß-cells within intact islets showed cell-specific timing of tolbutamide-induced response. NADH responses to glucose were cell-specific in all three mouse models, but the timing of NADH responses to KIC was cell-specific only in lean mice. Thus, a cell-specific response can be induced in normal ß-cells at several steps of stimulus-secretion coupling for nutrient-stimulated insulin release. Cell-specific properties of ß-cell ion channels and the mitochondrial metabolism are affected in db/db and ob/ob mice. The relation between mitochondrial mass and parameters of Ca2+ responses were investigated in Mitotracker Red and Fluo-3 labelled ß-cells using confocal microscopy. Data show that ß-cell mitochondrial state may play an important role in determining the timing of [Ca2+]i changes. In summary, the early Ca2+ response pattern in ß-cells, including the lag time, the nadir of initial lowering and the height of the first peak response is cell-specific. Isolated and functionally coupled ß-cells show cell-specific timing of Ca2+ responses when stimulated with metabolic and non-metabolic agents. This may be a robust mechanism of importance for the adequate function of ß-cells and a basis for the pacemaker function of some cells. A disturbed cell specificity of the mitochondrial metabolism and ion channel function appears to be a marker of ß-cell dysfunction in hyperglycemia and diabetes and may explain the delayed insulin release in ß-cells from diabetic subjects.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Neste estudo foram preparados polimorfos do fármaco tolbutamida, um hipoglicemiante oral usado no tratamento dos Diabetes Mellitus tipo II. Foram também preparados polimorfos da nifedipina, fármaco usado no tratamento das desordens cardiovasculares, como angina pectoris e hipertensão. A preparação dos polimorfos foi mediada por solvente, ou seja, foi em função do solvente usado nas etapas de cristalização e de precipitação das espécies. Um método de resfriamento rápido por nitrogênio líquido também foi utilizado. Técnicas analíticas como a espectrofotometria de infravermelho, a calorimetria diferencial de varredura, a difratometria de raio-X e a microscopia eletrônica de varredura foram úteis para a caracterização dos produtos obtidos experimentalmente. Os resultados comprovaram que dois polimorfos da tolbutamida foram preparados, ambos com estrutura cristalina. No caso da nifedipina, dois polimorfos foram preparados e a caracterização mostrou que um destes foi obtido num estado amorfo enquanto o outro estava sob forma cristalina. A instabilidade da nifedipina no estado amorfo foi monitorada pela técnica de calorimetria diferencial de varredura que, através de diferentes curvas, mostrou uma transformação rápida para uma estrutura cristalina. Esta mesma técnica aliada à termogravimetria confirmou a obtenção de um terceiro produto da nifedipina, de estrutura cristalina, que foi considerado um pseudopolimorfo por ser uma espécie solvatada. Ao final do procedimento experimental e da avaliação dos resultados foi sugerido um esquema, passo a passo, para obtenção e caracterização de polimorfos de uma substância / In this study the polymorphs of tolbutamide, an oral hypoglicemiant used on Diabetes Mellitus type II treatment, and of nifedipine, a drug used in the cardiovascular disorders treatment, were prepared. All crystalline forms were obtained by crystallization from different solvents. Tolbutamide was isolated only in crystalline forms and nifedipine in two crystalline forms and in the amorphous form prepared by melting and subsequent cooling. The polymorphs from each drug were characterized by powder x-ray diffraction (PDRX), infrared spectroscopy (IR), Raman spectroscopy (FT-RAMAN), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The results proved that two different crystalline forms of tolbutamide were obtained and two crystalline form to nifedipine, one of them as a pseudo-polymorph. The characterization confirmed that melting and quickly cooling procedure prepared amorphous nifedipine. Differential scanning calorimetry technique generated curves whose data proved that the amorphous nifedipine is a very unstable form. Thermogravimetry confirmed a pseudo-polymorphs preparation of nifedipine. In spite of the modification observed on the profile of X-ray diffraction, because of the solvent present, was possible to prove that this solvated form have an crystalline structure. A methodology was proposed step by step to prepare and characterize polymorphs of a substance

Hair disorders cause psychological distress but are generally poorly controlled; more effective treatments are required. Despite the long-standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP-sensitive potassium (K(ATP)) channels. To investigate whether K(ATP) channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tolbutamide, a K(ATP) channel blocker, opposed each other's effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase-polymerase chain reaction identified K(ATP) channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore-forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were examined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhibited SUR2B and Kir6.1. Immunohistochemistry demonstrated similar protein distributions. Thus, human follicles respond biologically to K(ATP) channel regulators in culture and express genes and proteins for two K(ATP) channels, Kir6.2/SUR1 and Kir6.1/SUR2B; minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain K(ATP) channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.

No description available.