Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells.

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells.

Isolation of chromaffin granules.

Adrenal medullary chromaffin granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of chromaffin granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified granules and granule membranes in a short period of time. Two protocols are presented here for the isolation of chromaffin granules: a basic protocol based on differential centrifugation in an iso-osmotic medium that yields intact chromaffin granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified chromaffin granules. Since in the latter case the granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the granule membranes or contents for further study.

Chromogranin A deficiency in transgenic mice leads to aberrant chromaffin granule biogenesis.

The biogenesis of dense-core secretory granules (DCGs), organelles responsible for the storage and secretion of neurotransmitters and neuropeptides in chromaffin cells, is poorly understood. Chromogranin A (CgA), which binds catecholamines for storage in the lumen of chromaffin granules, has been shown to be involved in DCG biogenesis in neuroendocrine PC12 cells. Here, we report that downregulation of CgA expression in vivo by expressing antisense RNA against CgA in transgenic mice led to a significant reduction in DCG formation in adrenal chromaffin cells. The number of DCGs formed in CgA antisense transgenic mice was directly correlated with the amount of CgA present in adrenal medulla. In addition, DCGs showed an increase in size, with enlargement in the volume around the dense core, a phenomenon that occurs to maintain constant "free" catecholamine concentration in the lumen of these granules. The extent of DCG swelling was inversely correlated with the number of DCGs formed, as well as the amount of CgA present in the adrenal glands of CgA antisense transgenic mice. These data indicate an essential role of CgA in regulating chromaffin DCG biogenesis and catecholamine storage in vivo.

Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog.

The secretory prohormone chromogranin A (CHGA) is overexpressed in essential hypertension, a complex trait with genetic predisposition, while its catecholamine release-inhibitory fragment catestatin is diminished, and low catestatin predicts augmented adrenergic pressor responses. These findings from studies on humans suggest a mechanism whereby diminished catestatin might increase the risk for hypertension. We generated Chga and humanized mice through transgenic insertion of a human CHGA haplotype in order to probe CHGA and catestatin in vivo. Chga mice displayed extreme phenotypic changes, including: (a) decreased chromaffin granule size and number; (b) elevated BP; (c) loss of diurnal BP variation; (d) increased left ventricular mass and cavity dimensions; (e) decreased adrenal catecholamine, neuropeptide Y (Npy), and ATP contents; (f) increased catecholamine/ATP ratio in the chromaffin granule; and (g) increased plasma catecholamine and Npy levels. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin replacement or humanization of Chga mice. Loss of the physiological "brake" catestatin in Chga mice coupled with dysregulation of transmitter storage and release may act in concert to alter autonomic control of the circulation in vivo, eventuating in hypertension.

Lack of the response to nicotine in 21-day-old rat adrenal chromaffin cells in vivo.

Response to nicotine of adrenal chromaffin cells was studied in suckling and young adult male rats in vivo. When 5 mg/kg of nicotine was injected subcutaneously to 8-week-old rats, the content of adrenaline and noradrenaline in the chromaffin granule fraction decreased about by 36 and 45%, respectively, 2 min after the administration. In electron microscopy, the number of chromaffin granules in the perinuclear region of adrenaline-storing cells decreased markedly. The number of vacuoles, probably produced by membrane recycling resulting from exocytosis, increased significantly in adrenaline- and noradrenaline-storing cells. Omega-shaped profiles (exocytosis) were frequently observed both in adrenaline- and noradrenaline-storing cells. On the other hand, nicotine injection did not significantly alter the catecholamine content in the 21-day-old rat chromaffin granule fraction, although severe convulsion was evoked. In electron microscopy, the changes indicative of exocytosis mentioned above were scarcely observed. Cholinergic nerve fibers of mature appearance were observed in the adrenal medulla of 21-day-old rats. These results indicate that the responsiveness of the chromaffin cells to nicotine in 21-day-old rats differs from that in 8-week-old rats.

Carcinoide apendicular

Desde noviembre de 1978 a junio de 1991 tratamos a 10 pacientes que presentaban un carcinoide de localización apendícular. Pertenecían al sexo masculino 6 pacientes y 4 al femenino, con una edad promedio de 42 años (rango de 20-68). En 9 pacientes se realizó una apendicectomía y en 1 una hemicolectomía derecha por otra patología colónica asociada. En ningún caso se realizó un diagnóstico preoperatorio y en 3 se sospechó patología apendícular durante otra intervención quirúrgica. En 7 casos el diagnóstico preoperatorio fue apendicitis aguda, sin embargo, el estudio anátomo-patológico no reveló inflamación apendícular en 5 casos. Ningún paciente presentó un síndrome carcinoide. Todos los tumores fueron menores 1 cm., en 9 casos se ubicaban en la punta y 1 en la base (a este último se le efectuó una hemicolectomía derecha por presentar una patología asociada). Pertenecían al tipo clásico 8 pacientes, y 2 al tipo tubular. No se registró ningún caso con células globosas, formas mixtas o asociadas con adenocarcinomas. El seguimiento se realizó durante una media de 7 años (rango de 1-14) sin registrar mortalidad. Consideramos importante para adecuar la terapéutica y el seguimiento tener en cuenta el tamaño la ubicación y la variedad histológica

Carcinoide apendicular

Desde noviembre de 1978 a junio de 1991 tratamos a 10 pacientes que presentaban un carcinoide de localización apendícular. Pertenecían al sexo masculino 6 pacientes y 4 al femenino, con una edad promedio de 42 años (rango de 20-68). En 9 pacientes se realizó una apendicectomía y en 1 una hemicolectomía derecha por otra patología colónica asociada. En ningún caso se realizó un diagnóstico preoperatorio y en 3 se sospechó patología apendícular durante otra intervención quirúrgica. En 7 casos el diagnóstico preoperatorio fue apendicitis aguda, sin embargo, el estudio anátomo-patológico no reveló inflamación apendícular en 5 casos. Ningún paciente presentó un síndrome carcinoide. Todos los tumores fueron menores 1 cm., en 9 casos se ubicaban en la punta y 1 en la base (a este último se le efectuó una hemicolectomía derecha por presentar una patología asociada). Pertenecían al tipo clásico 8 pacientes, y 2 al tipo tubular. No se registró ningún caso con células globosas, formas mixtas o asociadas con adenocarcinomas. El seguimiento se realizó durante una media de 7 años (rango de 1-14) sin registrar mortalidad. Consideramos importante para adecuar la terapéutica y el seguimiento tener en cuenta el tamaño la ubicación y la variedad histológica

Effects of the catecholaminergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) on adrenal chromaffin cells in culture.

N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is an inhibitor of noradrenaline uptake and a neurotoxin known to deplete noradrenaline levels with little effect on dopamine, serotonin or adrenaline in the central nervous system. The present study tested the effects of DSP-4 on catecholamine uptake, release and content in cultures of isolated bovine adrenal chromaffin cells. DSP-4 selectively inhibited the acute uptake of [3H]noradrenaline with little or no effect on [3H]adrenaline or [3H]dopamine uptake. In cultures preloaded with [3H]catecholamines, DSP-4 stimulated the release of [3H]noradrenaline and, to a small extent, also [3H]adrenaline and [3H]dopamine. However, the drug did not stimulate the release of appreciable amounts of endogenous adrenaline, noradrenaline or dopamine. A high concentration of DSP-4 inhibited the carbachol-stimulated release of adrenaline, noradrenaline and dopamine from the cells. Following a 1-hr exposure to the drug, DSP-4 decreased adrenaline, noradrenaline and dopamine levels in the cells with no gross morphologic changes in the cells. Reductions in adrenaline and noradrenaline levels were almost equal in magnitude, while dopamine was depleted to a somewhat greater extent under some conditions. Longer exposure to DSP-4 resulted in morphological changes in the cells, suggesting that the drug is also toxic to chromaffin cells in culture.

Impaired neurotransmitter uptake in PC12 cells overexpressing human Cu/Zn-superoxide dismutase--implication for gene dosage effects in Down syndrome.

Rat PC12 cells expressing elevated levels of transfected human Cu/Zn-superoxide dismutase (CuZn-SOD) gene were generated. These transformants (designated PC12-hSOD) closely resembled the parental cells in their morphology, growth rate, and response to nerve growth factor, but showed impaired neurotransmitter uptake. The lesion was localized to the chromaffin granule transport mechanism. We found that the pH gradient (delta pH) across the membrane, which is the main driving force for amine transport, was diminished in PC12-hSOD granules. These results show that elevation of CuZnSOD activity interferes with the transport of biogenic amines into chromaffin granules. Since neurotransmitter uptake plays an important role in many processes of the central nervous system, CuZnSOD gene-dosage may contribute to the neurobiological abnormalities of Down's syndrome.

Primary carcinoid of prostate.

The first case of pure primary prostatic carcinoid is presented. Previous demonstration of the presence of enterochromaffin cells in the non-neoplastic prostate and in otherwise ordinary adenocarcinomas of the prostate furnishes a basis for the origin of this unique neoplasm.