Nerve Growth Factor Effect on Adenosine Transport in Cultured Chromaffin Cells

Chromaffin cells both recently isolated or in culture present a high-affinity adenosine transporter with a Km value of 1 microM. When cells were exposed to nerve growth factor (NGF; 10 ng/ml), the adenosine transporter affinity decreased to 3 microM. This value was maintained from 3 days after plating to the end of the culture period. A change in the transport capacity was observed, with a significant increase (approximately 200-260%) in NGF-cultured cells throughout the period studied.     

Long term regulation of nucleoside transport by thyroid hormone (T3) in cultured chromaffin cells

The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-l-thyronine (T₃) throughout the prolonged period studied. The V_max values of this transport obtained in absence and presence of 1 M T₃ were 36.21±2.1 and 44.17±3.5 (means±SD) pmol/10⁶cells/min respectively for 26 hours incubation-time with the hormone. The K_m values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [³H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 M T₃ for 26 hours incubation-time. The values of binding sites per cell were 33,500±3,000 and 40,153±3,700 in absence and presence of T₃ respectively, without changing the K_d constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9±2.8 and 24.3±2.1 hours both in the presence or absence of T₃ respectively. When cells were incubated in the presence of both T₃ and cycloheximide, not only the activatory effect of T₃ was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T₃ activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism.     

Carbachol induced release of diadenosine polyphosphates--Ap4A and Ap5A--from perfused bovine adrenal medulla and isolated chromaffin cells.

The diadenosine polyphosphates--Ap4A and Ap5A--were released from perfused bovine adrenal glands and recently isolated chromaffin cells by the action of carbachol. The H.P.L.C. technique reported here allowed the quantification of pmol amounts of these compounds present in biological samples from the perfusion media after stimulation. Both compounds (Ap4A and Ap5A) were identified by the retention time in H.P.L.C. chromatography, co-elution with standards, re-chromatography and destruction by the phosphodiesterase action. Bovine adrenal glands stimulated with 100 microM carbachol released 0.47 +/- 0.12 nmol/gland of Ap4A and 1.11 +/- 0.26 nmol/gland of Ap5A. Isolated bovine chromaffin cells after 100 microM carbachol, as secretagogue, released 11.1 +/- 0.8 pmol/10(6) cells of Ap4A and 15.8 +/- 1.1 pmol/10(6) cells of Ap5A. The ratio of these compounds with respect to the exocytotically released ATP and catecholamines was in the same order as that found in isolated chromaffin granules.     

Two Components of Glutamate Exocytosis Differentially Affected by Presynaptic Modulation

Abstract: The total Ca²⁺-dependent release of glutamate induced by depolarization of cerebrocortical nerve terminals with KCl was analyzed into a fast and a slow component. The fast component exhibited a decay time of <1 s and accounted for 0.95 ± 0.10 nmol of glutamate, whereas the slow component, which exhibited a decay time of 52 ± 7 s, accounted for the release of 2.48 ± 0.19 nmol of glutamate. These two components were differentially affected by the Ca²⁺ chelator BAPTA, the divalent cation Sr²⁺, or the botulinum neurotoxin A. The adenosine A₁ receptor agonist N ⁶-cyclohexyladenosine strongly reduced the fast component without altering the slow component. In contrast, the inhibitory effect of arachidonic acid and the facilitatory action of the metabotropic glutamate receptor agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid were observed as a decrease and an increase, respectively, in the two components. It is concluded, first, that the fast and slow components correspond to the release of docked and mobilized vesicles, respectively, and second, that presynaptic modulation more significantly alters the fast component of release.     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis

Abstract: The effects of arachidonic acid and phorbol esters in the Ca²⁺-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 n M ) of 4β-phorbol dibutyrate (4β-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β-PDBu (1–50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.     

Nitrobenzylthioinosine binding cooperativity in chromaffin tissue membranes.

The nucleoside transporter present in chromaffin tissue membranes has been studied by [3H]nitrobenzylthioinosine (NBTI) binding. This ligand presents a high affinity, with a Kd value of 2.1 +/- 0.2 nM and a Bmax of 1.7 +/- 0.2 pmol/mg protein. From the Scatchard and the semilogarithmic graphical representations a positive cooperativity was deduced, with a Hill coefficient of 1.7 +/- 0.4. In displacement studies of NBTI by the non labelled compound, the Hill coefficient was also higher than 1 (1.44 +/- 0.11) in the presence of ATP. This nucleotide seems necessary to maintain the number of high affinity binding sites.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.