Transplantation of microencapsulated bovine chromaffin cells reduces lesion-induced rotational asymmetry in rats

Surrounding bovine chromaffin cells by a semipermeable membrane may protect the transplanted cells from a host immune response and shield them from the inflammatory process resulting from the surgical trauma. Encapsulation of the chromaffin cells was achieved by inter-facial adsorption of a polycation on a polyanionic colloid matrix in which the chromaffin cells were entrapped. Basal and potassium-evoked release of catecholamines from encapsulated bovine chromaffin cells was analyzed over a 4-week period in vitro. Norepinephrine and dopamine release remained constant over time whereas epinephrine release significantly decreased. The chromaffin cells also retained the capacity for depolarization-elicited catecholamine release 4 weeks following the encapsulation procedure. Morphological analysis revealed the presence of intact chromaffin cells with well-preserved secretory granules. Striatial implantation of chromaffin cell-loaded capsules significantly reduced apomorphine-induced rotation compared to empty polymer capsules in animals lesioned with 6-hydroxydopamne frr at least 4 weeks. Intact chromaffin cells expressing tyrosine hydroxylase and dopamine-β-hydroxylase were observed in all capsules implanted in the striatum for 4 weeks. The assessment of the clinical potential of transplanting encapsulated adrenal chromaffin cells of either allo- or xenogeneic origin for Parkinson's disease will require long-term behavioral studies. The present study suggests, however, that the polymer encapsulation procedure may offer an alternative to adrenal autografts as a source of dopaminergic tissue.     

The multifunctionality of FGF-2 in the adrenal medulla

 Chromaffin cells of the adrenal medulla and their tumor counterparts, the pheochromocytoma (PC12) cells, are well-established model systems in neurobiology. The development of sympathoadrenal progenitor cells to chromaffin cells can be studied with regard to developmental signals which trigger the differentiation. With regard to potential treatments of neurological disorders like Parkinson’s disease chromaffin cell grafting can be used as one therapeutical approach. The beneficial effect of chromaffin cell grafts is possibly not only related to the release of dopamine but may also be linked to the release of growth factors. One of the growth factors that is synthesized by chromaffin and PC12 cells is basic fibroblast growth factor (FGF-2). The experimental data available so far, are in agreement with different functional roles of FGF-2. This article summarizes the putative physiological functions of FGF-2 in the adrenal medulla. Three differential functional roles of FGF-2 are discussed: (1) as a differentiation factor for sympathoadrenal progenitor cells; (2) as a target-derived neurotrophic factor for preganglionic sympathetic neurons which innervate adrenal medullary cells; (3) as an auto-/paracrine factor in the adrenal medulla. Accepted: 21 August 1996     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Modeling of stimulation–secretion coupling in a chromaffin cell

We constructed a chromaffin cell model for analysis of stimulation–secretion coupling in computer simulation studies. The model includes mechanisms involved in the excitatory synapse, voltage-dependent Na⁺, K⁺ and Ca²⁺ channels, Ca²⁺-activated K⁺ channels (SK type), buffered Ca²⁺ diffusion, Ca²⁺ extrusion, fluorescent Ca²⁺ indicators and Ca²⁺-triggered exocytosis. Calculations of the modeled mechanisms were carried out using the NEURON simulation environment (Hines and Carnevale, Neural Computation 9:1179–1209, 1997). A set of parameter values was determined so as to fit basic experimental results reported in the literature. The model was also applied to simulate our experimental results obtained from chromaffin cells in the perfused rat adrenal medulla. Observed profiles of Ca²⁺responses induced by electrically stimulating the splanchnic nerve with various frequencies (1–50 Hz) were adequately simulated with minor readjustments of parameter values for Ca²⁺influx and extrusion. Secretory responses measured at the same time as the Ca²⁺responses were also simulated with consideration of a time constant to detect catecholamines in the experiment. Similarly, model simulations reproduced both Ca²⁺responses and secretory responses evoked by elevations of the extracellular K⁺ concentration for different periods. The results suggest that the presented model provides a useful tool for analyzing and predicting quantitative relations in various events occurring in stimulation–secretion coupling in chromaffin cells.     

Evidence for an inherent,ATP-stimulated uptake of calcium into chromaffin granules

Summary Partially purified chromaffin granules (granular fraction), crude mitochondria (mitochondrial fraction) and a microsomal fraction were prepared from bovine adrenals by differential centrifugation and characterized by their catecholamine content, succinate dehydrogenase and glucose-6-phosphatase activity. During isotonic incubation with 0.1 mM ⁴⁵Ca²⁺ all fractions showed an uptake of ⁴⁵Ca²⁺, which was stimulated by ATP. In addition, after incubation the granular fraction was further fractionated by sucrose density gradient centrifugation (2.0–1.3 M sucrose; 170,000g·60 min). In some of these experiments the granular fraction was incubated simultaneously with ⁴⁵Ca²⁺ and [³H](-)noradrenaline to compare the uptake of both. The rate of uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction (characterized by the highest catecholamine content and the lowest succinate dehydrogenase activity) was doubled by ATP. The ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules was inhibited by N-ethylmaleimide (NEM) (0.1 mM), 2,4-dinitrophenol (DNP) (0.1 mM), azide (1 mM), carbonyl cyanide mchlorophenylhydrazone (CCCP) (20M), atractyloside (50 M), ruthenium red (40 M) and amobarbital (1 mM). This inhibition pattern was different from that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the microsomal fraction, but it was similar to that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction. However, the following differences are incompatible with the view that a mitochondrial contamination, with a highly active uptake, is responsible for the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules: a) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was insensitive to 1 mM amobarbital, whereas this agent inhibited the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules. b) Replacement of ATP by succinate stimulated the uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction only. c) The dependence of the ATP-stimulated uptake of ⁴⁵Ca²⁺ on the concentration of ATP (0.1–5 mM) was determined: while uptake into the 2.0 M sucrose fraction of chromaffin granules exhibited saturation kinetics, that into the mitochondrial fraction was linearly related to the concentration of ATP. Interestingly, uptake of ⁴⁵Ca²⁺ into those fractions of chromaffin granules that are known to be contaminated with mitochondria (1.6 M sucrose fraction) exhibited bot a saturable and a nonsaturable component. d) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was more sensitive to 0.1 mM DNP than the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules.The comparison of the ATP-stimulated uptake of ⁴⁵Ca²⁺ with that of [³H](-)noradrenaline into the 2.0 M sucrose fraction of chromaffin granules revealed that amobarbital, N,N-dicyclohexylcarbodiimide (DCCD), DNP and increasing concentrations of ATP had the same influence on both uptake processes. From our results we conclude that an inherent, ATP-stimulated uptake of ⁴⁵Ca²⁺ exists in chromaffin granules. The effects of agents on the uptake of ⁴⁵Ca²⁺ and/or [³H](-)noradrenaline into chromaffin granules are discussed with regard to the granular uptake mechanisms.This work was supported by the Deutsche ForschungsgemeinschaftSome results were reported at the 18th Spring Meeting 1977 of the Deutsche Pharmakologische Gesellschaft (Burger and Häusler 1977)     

Neural tissue engineering: Adrenal chromaffin cell attachment and viability on chitosan scaffolds

Abstract

This study introduces chitosan-based matrices as cell substrates for bovine chromaffin cell attachment in transplantation procedures. Chitosan ([1 → ] linked 2-amino-2-deoxy-β-O-glucopyranose), having structural similarity to glycosaminoglycans, was modified using several proteins (collagen, albumin and gelatin) to increase surface area and improve biocompatibility. In vitro, collagen-blended chitosan (CC) matrices were found to attach more readily to chromaffin cells than to gelatin- or albumin-blended matrices. Morphological evidence showed that the chromaffin cells attached to CC substrates integrated well with the hydrogel matrix and survived for at least two weeks, under in vivo culture conditions. The chromaffin cells within chitosan scaffolds also survived for at least two weeks in vitro and after subarachnoid grafting to rats. [Neural Res 1998; 20: 648–654]     

糖皮质激素对大鼠肾上腺嗜铬细胞分泌儿茶酚胺的急性效应

利用全细胞膜片钳技术,观察不同浓度地塞米松急性灌洗大鼠肾上腺嗜铬细胞(AMCC)后钙通道电流和烟碱受体通道电流(INIC)的变化,地塞米松对大鼠AMCC的急性作用为明显抑制INIC而对电刺激所诱发的钙通道电流无明显影响,提示糖皮质激素对大鼠AMCC分泌儿茶酚胺的急性效应可能与烟碱受体直接相关。     

Sympathetic nervous system activation and heart failure: Current state of evidence and the pathophysiology in the light of novel biomarkers

Heart failure (HF) is a complex clinical syndrome characterized by the activation of at least several neurohumoral pathways that have a common role in maintaining cardiac output and adequate perfusion pressure of target organs and tissues. The sympathetic nervous system (SNS) is upregulated in HF as evident in dysfunctional baroreceptor and chemoreceptor reflexes, circulating and neuronal catecholamine spillover, attenuated parasympathetic response, and augmented sympathetic outflow to the heart, kidneys and skeletal muscles. When these sympathoexcitatory effects on the cardiovascular system are sustained chronically they initiate the vicious circle of HF progression and become associated with cardiomyocyte apoptosis, maladaptive ventricular and vascular remodeling, arrhythmogenesis, and poor prognosis in patients with HF. These detrimental effects of SNS activity on outcomes in HF warrant adequate diagnostic and treatment modalities. Therefore, this review summarizes basic physiological concepts about the interaction of SNS with the cardiovascular system and highlights key pathophysiological mechanisms of SNS derangement in HF. Finally, special emphasis in this review is placed on the integrative and up-to-date overview of diagnostic modalities such as SNS imaging methods and novel laboratory biomarkers that could aid in the assessment of the degree of SNS activation and provide reliable prognostic information among patients with HF.     

Improved neural progenitor cell survival when cografted with chromaffin cells in the rat striatum

Transplantation of stem and neural progenitor cells hold great promise in the repair of neuronal tissue lost due to injury or disease. However, survival following transplantation to the adult CNS has been poor, likely due to a lack of neurotrophic factors, such as basic fibroblast growth factor (FGF-2), that are used to maintain and expand these cells in culture. Chromaffin cells produce several neurotrophic agents, including FGF-2, which may aid in both neuroprotection following injury and progenitor cell proliferation and survival. In addition, increased CNS catecholamines have been shown to improve functional recovery following insult. Thus, cotransplants of neural progenitor cells and chromaffin cells may be a useful clinical strategy. To address this, the survival of rat cortical progenitors transplanted to the adult rat striatum with and without bovine chromaffin cell cografts was assessed. Progenitors obtained from E14 embryos were prelabeled with bromodeoxyuridine (BrdU) before transplantation to enable later identification. Transplants were made both unilaterally and bilaterally, where animals received a monograft (progenitor cells alone) on one side and a cograft (progenitors + chromaffin cells) on the other. Histological results after 7, 17, and 30 days posttransplant revealed greatly improved survival of BrdU-labeled cells in the cografts and also less infiltration of presumptive immune cells. In addition, perivascular cuffing was seen in the monografts. In vitro progenitor cohorts stained positive for nestin, GFAP, and beta-tubulin III, but in vivo very few cells were found that were double labeled with BrdU and one of these markers. Thus, in contrast to in vitro findings, chromaffin cells did not enhance differentiation of progenitors in vivo during the 30 days posttransplantation. The results of these studies suggest that chromaffin cells may provide neurotrophic support to enhance survival, but not differentiation, of cortical progenitor grafts in the adult CNS.