Adenosine triphosphate in the bovine chromaffin granule.

1. pH and potential gradients are generated across the membranes of chromaffin granule 'ghost' by incubating them with MgATP: the inside of the 'ghosts' is positive and acid with respect to the incubation medium. 2. The pH gradient is partially dissipated by inclusion of a substrate for the catecholamine pump, or a mitochondrial uncoupling agent, but is enhanced by reserpine. 3. An imposed pH gradient leads to amine uptake by the 'ghosts': a potential gradient leads to ATP uptake. Studies with inhibitors confirm that amine accumulation by chromaffin granules is dependent on the former, and that ATP uptake results from ATPase-induced potential difference generation. 4. ATP has two known roles in chromaffin granule structure: the first is as a substrate for a membrane-bound proton-translocating ATPase; the second is as a component of the intragranular catecholamine storage complex.     

Effects of cytoskeleton-disrupting agents on tyrosine transport into cultured bovine adrenal chromaffin cells

The effects of various cytoskeleton-disrupting agents on tyrosine transport into chromaffin cells were examined to assess the possibility of cytoskeleton involvement in the regulation of precursor supply for catecholamine synthesis. Tyrosine transport was markedly increased by cytochalasin B. Vinblastine also stimulated tyrosine transport, although its effect was less pronounced than that of cytochalasin B. While colchicine failed to cause any significant increase in the transport under the same conditions. These results therefore suggest a possible role of microfilaments as a factor regulating tyrosine transport into chromaffin cells.     

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.     

Phosphoglyceride biosynthesis in bovine adrenal chromaffin cells

Cultured adrenal chromaffin cells, representing a virtually homogeneous population of neuronai elements, have been utilized to examine the final enzymes in the formation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), namely, choline phosphotransferase, ethanolaminephosphotransferase, and the N-methyltransferases in the sequential methylation of PE to PC. Each enzyme has been characterized extensively in terms of substrate requirements, pH optima, detergent and cation effects, and response to inhibitors revealing properties very similar to those in other neural preparations. The respective activities are stable for up to two weeks of adrenal chromaffin cell culture suggesting that this system is a suitable model for examining the relative roles and the regulation of each pathway in PC formation.Abbreviations EPT ethanolaminephosphotransferase - CPT cholinephosphotransferase - NMT N-methyltransferase This work supported by funds provided to the Section of Pediatric Neurology by Texas Children's Hospital.     

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

• 1.1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture.
• 2.2. Specific ¹²⁵I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K_d~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing ¹²⁵I-labelled insulin.
• 3.3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of ¹²⁵I-labelled insulin and ¹²⁵I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types.
• 4.4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Adenosine transporters in chromaffin cells. Quantification by dipyridamol monoacetate

Chromaffin cells from bovine adrenal medulla are a useful model to approach adenosine transport and metabolism in neural cells. Dipyridamol has been shown to be an adenosine transport inhibitor with high affinity. To quantify the adenosine transporters a labelled dipyridamol analogue, [14C]dipyridamol acetate, was synthesized. This compound had a Ki = 5.3 +/- 0.43 nM according to the Dixon method, and 4.58 +/- 0.46 nM when the receptor number molarity was taken into account showing, like dipyridamol, a non-competitive mechanism. The high-affinity receptors present in chromaffin cells showed a Kd = 6.8 +/- 0.8 nM and the receptor number was 630 000 +/- 40 000 per cell.     

Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture.

An ectoenzyme hydrolyzing diadenosine polyphosphates (ApnA) to AMP and Ap(n-1) has been studied in cultured chromaffin cells from bovine adrenal medulla. The KM value for extracellular Ap4A hydrolysis was 2.90 +/- 0.72 microM, the V(max) value obtained was 11.59 +/- 0.92 pmol/min x 10(6) cells (116 pmol/min.mg total protein). Ap3A, Ap5A, Ap6A, and Gp4G were competitive inhibitors of Ap4A hydrolysis with K(i) values of 3.65, 1.10, 1.20, and 2.65 microM, respectively. Phosphatidylinositol-specific phospholipase C removes the ApnA hydrolase activity from cultured chromaffin cells, suggesting an anchorage of this protein to the plasma membrane through the phosphatidylinositol. The turnover time for this enzyme calculated in the presence of cycloheximide was 38.94 +/- 1.53 hr for cultured chromaffin cells.     

GLUT1-mediated glucose transport and its regulation by IGF-I in cultured bovine chromaffin cells

Regulation of glucose transport was studied in primary cultures of bovine chromaffin cells (BCC) using the glucose analogue 2-deoxyglucose (DOG) as a model substrate. The glucose transporter in freshly isolated and cultured BCC was identified as GLUT1 by Western immunoblots. The level of GLUT1 increased by time in culture and was followed by an enhancement in uptake of DOG. The DOG uptake was stimulated by insulin-like growth factor I (IGF-I) with an EC₅₀ of 1 nM and a maximal response (∼2-fold) was obtained at 10–100 nM IGF-I. Insulin was at least 100-fold less potent than IGF-I. Exposure to 10⁻⁸ M IGF-I also caused a redistribution of GLUT1 from an intracellular compartment to a plasma membrane-enriched fraction. Our results demonstrate a GLUT1-mediated glucose uptake in adrenomedullary cells. An enhanced glucose transport in response to IGF-I appears to be coupled to activation of IGF receptor type 1 and GLUT1 translocation. © 1996 Wiley-Liss, Inc.     

Calcium-dependent transferrin receptor recycling in bovine chromaffin cells

The release of regulated secretory granules is known to be calcium dependent. To examine the Ca²⁺-dependence of other exocytic fusion events, transferrin recycling in bovine chromaffin cells was examined. Internalised ¹²⁵I-transferrin was released constitutively from cells with a half-time of about 7 min. Secretagogues that triggered catecholamine secretion doubled the rate of ¹²⁵I-transferrin release, the time courses of the two triggered secretory responses being similar. The triggered ¹²⁵I-transferrin release came from recycling endosomes rather than from sorting endosomes or a triggered secretory vesicle pool. Triggered ¹²⁵I-transferrin release, like catecholamine secretion from the same cells, was calcium dependent but the affinities for calcium were very different. The extracellular calcium concentrations that gave rise to half-maximal evoked secretion were 0.1 m m for ¹²⁵I-transferrin and 1.0 m m for catecholamine, and the intracellular concentrations were 0.1 μ m and 1 μ m , respectively. There was significant ¹²⁵I-transferrin recycling in the virtual absence of intracellular Ca²⁺, but the rate increased when Ca²⁺ was raised above 1 n m , and peaked at 1 μ m when the rate had doubled. Botulinum toxin type D blocked both transferrin recycling and catecholamine secretion. These results indicate that a major component of the vesicular transport required for the constitutive recycling of transferrin in quiescent cells is calcium dependent and thus under physiological control, and also that some of the molecular machinery involved in transferrin recycling/fusion processes is shared with that for triggered neurosecretion.     

Calcium gradients and exocytosis in bovine adrenal chromaffin cells

The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small "synchronous" increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.     

Calcium export by sodium-calcium exchange in bovine chromaffin cells

Calcium efflux from bovine chromaffin cells in tissue culture has been examined after loading them with small amounts of Ca2+ by brief depolarization in media containing 20 mumol/l to 1 mmol/l Ca2+ and 45Ca2+ in trace amounts. In the presence of normal external Na+ and Ca2+ concentrations cells depolarized in media containing up to 200 mumol/l Ca2+ exported nearly 100% of their accumulated Ca2+ loads within 10 min and 20% within the first 5 s. In the absence of external Na+ and Ca2+ the proportion of a small (i.e., depolarization in 20 mumol/l calcium) Ca2+ load exported at any time point in the range to 10 min was approximately two thirds of the total efflux measured in their presence indicating that under these conditions the external Na+/Ca(2+)-dependent and Na+/Ca(2+)-independent mechanisms both contribute significantly to the export of calcium. At higher cellular loads of calcium (i.e., depolarization in 200 mumol/l to 1 mmol/l calcium) the Na+/Ca(2+)-dependent mechanism exported a progressively greater proportion of the accumulated Ca2+. Both sodium and calcium alone promoted a component of Ca2+ efflux; Ca2+ (i.e. calcium-calcium exchange) was as effective as Na+ (i.e. sodium-calcium exchange). The Km for Na+ stimulation of Ca(2+)-efflux (KNa) was approximately 65 mM. Increased external Mg2+ (from 1.2 to 10 mmol/l) increased the apparent KNa to 90 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells

Chromaffin cells from bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: K(m) 90 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K(m) 52 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH range 7. 4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 microg of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg(2+)-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture (<4%; still moderate inhibition at more elevated concentrations). The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed.     

3-O-methyl-D-glucose uptake in cultured bovine adrenal chromaffin cells

The membrane transport of glucose was studied in bovine adrenal chromaffin cell cultures by following the cell/medium distribution of the nonmetabolizable glucose analog, 3-O-methyl-D-glucose. Uptake of this sugar in day-1 cultures that are undergoing rapid morphological change and differentiation had a Vmax of 138 nmol/(mg protein.min) and Km of 15 mM, and was only slightly increased by 50 mU/mL insulin. In day-5 cultures where morphological changes were essentially completed, Vmax and Km decreased to 51 nmol/(mg protein.min) and 9.5 mM, respectively, and the response to insulin was restored to the level found in freshly isolated cells; this effect was abolished in the nominal absence of Ca2+. Thus, saturation kinetics and insulin and Ca2+ sensitivity of 3-methylglucose uptake observed in freshly isolated cells were maintained in culture. However, the insulin response was almost absent during the initial period of rapid morphological change when sugar transport was strongly stimulated. Culture of chromaffin cells in the presence of dexamethasone did not inhibit the formation of processes, but decreased 3-methylglucose uptake in day-5 cultures by an apparently competitive effect.