Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo.
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Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for several populations of CNS and peripheral neurons. Synthesis and storage of GDNF by the neuron-like adrenal medullary cells suggest roles in adrenal functions and/or in the maintenance of spinal cord neurons that innervate the adrenal medulla. We show that unilateral adrenomedullectomy causes degeneration of all sympathetic preganglionic neurons within the intermediolateral column (IML) of spinal cord segments T7-T10 that project to the adrenal medulla. In situ hybridization revealed that IML neurons express the glycosylphosphatidylinositol-linked alpha receptor 1 and c-Ret receptors, which are essential for GDNF signaling. IML neurons also display immunoreactivity for transforming growth factor-beta (TGF-beta) receptor II. Administration of GDNF (recombinant human, 1 microg) in Gelfoam implanted into the medullectomized adrenal gland rescued all Fluoro-Gold-labeled preganglionic neurons projecting to the adrenal medulla after four weeks. Cytochrome c applied as a control protein was not effective. The protective effect of GDNF was prevented by co-administration to the Gelfoam of neutralizing antibodies recognizing all three TGF-beta isoforms but not GDNF. This suggests that the presence of endogenous TGF-beta was essential for permitting a neurotrophic effect of GDNF. Our data indicate that GDNF has a capacity to protect a population of autonomic spinal cord neurons from target-deprived cell death. Furthermore, our results demonstrate for the first time that the previously reported requirement of TGF-beta for permitting trophic actions of GDNF in vitro (Kreiglstein et al., 1998) also applies to the in vivo situation. (+info)
Voltage inactivation of Ca2+ entry and secretion associated with N- and P/Q-type but not L-type Ca2+ channels of bovine chromaffin cells.
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1. In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high-voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2. The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+-free solution containing 1.2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0.65 +/- 0.02 fmol cell-1; in depolarized cells (bathed in nominally Ca2+-free solution containing 100 mM K+) the net Ca2+ uptake was 0.16 +/- 0.01 fmol cell-1. 3. This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura-2-loaded single cells, from 1181 +/- 104 nM in hyperpolarized cells to 115 +/- 9 nM in depolarized cells. 4. A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1.2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 +/- 35 nA of catecholamines in hyperpolarized cells and 220 +/- 19 nA in depolarized cells. 5. The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (2 microM), thus suggesting that depolarization caused a selective inactivation of the N- and P/Q-type Ca2+ channels. 6. This was strengthened by two additional findings: (i) nifedipine (3 microM), an L-type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 microM), an L-type Ca2+ channel 'activator', dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7. In voltage-clamped cells, switching the holding potential from -80 to -40 mV promoted the loss of 80 % of the whole-cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 microM nifedipine and dramatically enhanced by 3 microM FPL64176. 8. Thus, it seems that the N- and P/Q-subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L-subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions. (+info)
L- and T-type voltage-gated Ca2+ currents in adrenal medulla endothelial cells.
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We investigated voltage-dependent Ca2+ channels of bovine adrenal medulla endothelial cells with the whole cell version of the patch-clamp technique. Depolarization elicited an inward current that was carried by Ca2+ and was composed of a transient (T) current, present in all the cells tested, and a sustained (L) current, present in 65% of them. We separated these currents and measured their individual kinetic and gating properties. The activation threshold for T current was approximately -50 mV, and its maximum amplitude was -49.8 +/- 4.8 pA (means +/- SE, n = 19) at 0 mV. The time constant was 10.2 +/- 1.5 ms (n = 4) for activation and 18.4 +/- 2.8 ms (n = 4) for inactivation. The L current activated at -40 mV, and it reached a plateau at -20.1 +/- 2.3 pA (n = 6). Its activation time course was a single exponential with an activation time contant of 26.8 +/- 2.3 ms (n = 4). Current-voltage curves, kinetics, gating, response to BAY K 8644, nifedipine, amiloride, and different selectivity for Ba2+ and Ca2+ indicated that the underlying channels for the observed currents are only of the T- and L-types that resemble those of the endocrine secretory cells. (+info)
Lambert-Eaton antibodies inhibit Ca2+ currents but paradoxically increase exocytosis during stimulus trains in bovine adrenal chromaffin cells.
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Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that affects neurotransmitter release at peripheral synapses. LEMS antibodies inhibit Ca2+ currents in excitable cells, but it is not known whether there are additional effects on stimulus-secretion coupling. The effect of LEMS antibodies on Ca2+ currents and exocytosis was studied in bovine adrenal chromaffin cells using whole-cell voltage clamp in perforated-patch recordings. Purified LEMS IgGs from five patients inhibited N- and P/Q-type Ca2+ current components to different extents. The reduction in Ca2+ current resulted in smaller exocytotic responses to single depolarizing pulses, but the normal relationship between integrated Ca2+ entry and exocytosis (Enisch and Nowycky, 1996) was preserved. The hallmark of LEMS is a large potentiation of neuromuscular transmission after high-frequency stimulation. In chromaffin cells, stimulus trains can induce activity-dependent enhancement of the Ca2+-exocytosis relationship. Enhancement during trains occurs most frequently when pulses are brief and evoke very small amounts of Ca2+ entry (Engisch et al., 1997). LEMS antibody treatment increased the percentage of trains eliciting enhancement through two mechanisms: (1) by reducing Ca2+ entry and (2) through a Ca2+-independent effect on the process of enhancement. This leads to a paradoxical increase in the amount of exocytosis during stimulus trains, despite inhibition of Ca2+ currents. (+info)
Studies on cyclic nucleotides in the adrenal gland. V. Adenylate cyclase in the adrenal medulla.
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Effects of various chemical agents eliciting the catecholamine-release on the adenylate cyclase-cyclic AMP generating system have been studied in the secretory process of the bovine adrenal medulla slices. Cyclic AMP levels were not affected at the interval of the maximal increase of the catecholamine-release by acetylcholine, but increased gradually some time after the end of the release/or at the beginning of the restoration of catecholamine in the medulla tissue. This delayed increase in the medullary cyclic AMP is not attributed to a direct involvement in 'stimulus-secretion coupling process' of the medullary secretion, but rather may be caused by release of intracellular catecholamine. (+info)
Regulation of basal expression of catecholamine-synthesizing enzyme genes by PACAP.
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We have previously reported that the cAMP/protein kinase A (PKA) pathway is important in the gene regulation of both induction and basal expressions of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to activate the intracellular cAMP/PKA pathway. In the present study, using primary cultured bovine adrenal medullary cells, we determined whether the basal activity of the PACAP receptor might play a role in the maintenance of the basal expression of these enzyme genes via the cAMP/PKA pathway. The potent PACAP receptor antagonist PACAP (6-38) caused a reduction of TH and DBH mRNA levels in a dose dependent manner as well as their enzyme activities and TH protein level. The effects of PACAP (6-38) and the PKA inhibitor H-89 exhibited generally similar trends, and were not additive in the reduction of TH and DBH gene expression and activities, suggesting that they take a common intracellular signaling pathway. The antagonist also caused decreases in the intracellular norepinephrine and epinephrine levels similar to the effect of H-89. Taken together, the data suggests that PACAP is involved in the regulation of maintenance of the catecholamine synthesizing enzymes TH and DBH by utilizing the cAMP/PKA pathway. (+info)
Electrical excitability of cultured adrenal chromaffin cells.
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1. Adult human and gerbil adrenal medullary cells were maintained in dissociated cell culture and studied by micro-electrode penetration. 2. In the best recordings, chromaffin cell transmembrane potentials exceeded -50mV. 3. Chromaffin cells were capable of generating all-or-nothing over-shooting action potentials, similar to those generated by sympathetic neurones. 4. The action potentials were blocked by tetrodotoxin (TTX, 10(-6)g/ml.) but were not blocked by removal of Ca or by CoCl2 (10 mM). We conclude that the action potentials are probably generated by a Na mechanism. 5. Chromaffin cells are depolarized by the iontophoretic application of acetylcholine (ACh). This depolarization was accompanied by an increased membrane conductance and could trigger action potentials. 6. Action potentials were also found in cells in fresh slices of gerbil adrenal medullae. (+info)
Influences of long-term administration of 24R, 25-dihydroxyvitamin D3, a vitamin D3 derivative, in rats.
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In order to examine the influences by long-term feeding of 24R, 25 dihydroxyvitamin D3[24R, 25(OH)2D3], an active form of vitamin D, Wistar rats (14-week-old, male, 20 rats/group) were fed a powder diet containing 0 or 5 ppm 24R, 25(OH)2D3 for 57 weeks. Final body weights and total food consumption were comparable between the groups. Urinary calcium levels were significantly (p < 0.05 or 0.01) increased by the administration of 24R, 25(OH)2D3 at weeks 3, 22 and 56, although the levels of serum calcium did not differ between the groups at the termination of week 57. In the 24R, 25(OH)2D3 group, weights of the adrenals and femurs were significantly (p < 0.01) increased. Histopathologically, this was found due to thickening of cortical bone in the femurs, and medullary hyperplasia and pheochromocytoma of the adrenals. Immunohistochemically, proliferating cell nuclear antigen (PCNA)-labeling indices for intact adrenal medulla, medullary hyperplasia and pheochromocytoma in the 24R, 25(OH)2D3 group were respectively 1.82 +/- 1.21, 5.88 +/- 4.13 and 16, all higher than that for the adrenal medulla in the control group (0.87 +/- 0.67). These results indicate that 24R, 25(OH)2D3 at a dose with which serum calcium is not chronically increased causes thickening of the cortex of the femur, and development of adrenal proliferative lesions, suggesting that rats may be too sensitive for results to be relevant to human risk assessment. (+info)