Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

α- and β-receptor control of catecholamine secretion from isolated adrenal medulla cells

Summary The structural characteristics and cellular elements of the boundary zone between the white and red pulp of the human spleen were studied by SEM and TEM. The boundary zone consisted of both the perifollicular region and the region surrounding the periarterial lymphoid sheath. The perifollicular region was further subdivided into two, equally thick layers. The inner half layer of the perifollicular region outside the mantle zone of the lymph follicle was composed of tightly packed medium-sized lymphocytes, interspersed by a small number of reticular cells. The outer half layer was composed of a reticular cell meshwork containing blood cells in vessels, which communicated with the splenic cords of the red pulp. Intermittent rows of reticular cells distinguished the outer from the inner half layer. The region surrounding the periarterial lymphoid sheath revealed the same type of reticular cell meshwork as the outer half layer of the perifollicular region. Capillary ends opened into the reticular cell meshwork, which suggested the presence of an open circulation in the human spleen. A deep lymphatic vessel which communicated with the periarterial lymphoid sheath was noted.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Membrane dopamine β-hydroxylase: a precursor for the soluble enzyme in the bovine adrenal medulla

• 1.1. Searching for endogenous proteolytic activities converting the membrane form of dopamine β-hydroxylase (dopamine β-monooxygenase, DBH) into the soluble and releasable form, DBH was monitored enzymatically and immunologically in aqueous and detergent-solubilized extracts of the adrenomedullary fractions.
• 2.2. Degradation of the soluble DBH and acidic chromogranins by activation of endogenous proteases occurred during lysis in H₂O.
• 3.3. Shifts in the hydrophobicity of the membrane DBH were also apparent. Loss in enzyme protein or activity was, on the other hand, not observed for bufier-dialysed CG (pH 5–6).
• 4.4. Limited proteolysis within the membrane phase was, however, indicated by the shift towards dominance of the intermediate hydrophobic DBH in the buffer-dialysed CG.
• 5.5. By two-dimensional, crossed immunoelectrophoresis with cationic detergent the microsomal DBH was immunologically identical to the granule-bound enzyme but differed from the latter in molecular heterogeneity and in susceptibility to proteolytic solubilization by endogenous protease activities.
• 6.6. DBH in the membranes of the chromaffin granules was proteolytically solubilized at pH 6–8 and the soluble DBH further degraded at pH 5.
• 7.7. The results indicate that a post-translational conversion of the amphiphilic DBH into the soluble form, initiated at the level of the microsomes, may continue within the light and the heavy granule fractions which contain several DBH-converting and degrading proteolytic activities with acid optima.

     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.     

Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

Appearance of tyrosine hydroxylase,aromatic amino-acid decarboxylase,dopamine β-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla

Summary The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 m in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls.In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline-(A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical anlage. PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the medullary cells have reached the cortical anlage. On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.This study was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) and by the Swedish Medical Research Council (04X-2887-10C). Its results have in part been reported at the 105th Meeting of the Dutch Anatomical Society (Abstract: Acta morphologica neerlando-scandinavica, 14, 251, 1976)     

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.     

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing.     

Involvement of proteases in glycosyltransferase secretion: Alzheimer's β-secretase-dependent cleavage and a following processing by an aminopeptidase

Alzheimer's beta-secretase (BACE1) cleaves amyloid precursor protein to produce amyloid beta-peptide, which is a crucial initiation process of the pathogenesis of Alzheimer's disease. We previously found that BACE1 also cleaves a membrane-bound sialyltransferase (ST6Gal I). Here we report that, when the protein A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, was used as an in vitro substrate for BACE1, it cleaved the substrates between Leu(37) and Gln(38). However, a soluble form of ST6Gal I secreted from COS cells started from Glu(41), which was three amino acids shorter than the in vitro product. The results suggested that the BACE1 product was truncated by an aminopeptidase(s) before secretion. The aminopeptidase activity was successfully detected in detergent extracts of Golgi-membrane fraction. Taken together, we concluded that BACE1 initially cleaved ST6Gal I between Leu(37) and Gln(38), and the NH(2)-terminal three amino acids of the yielded product was further trimmed by the aminopeptidase.     

Plasma dopamine β-hydroxylase species dependence and the in vitro influence of NEM,copper, and pH

We have applied a sensitive radiochemical assay to measure DBH activities in several species at their pH optima and to estimate the effects of copper and N-ethylmaleimide (NEM) in the assay. The enzyme incubation was performed at 37° with 5 μl of diluted plasma and 5 × 10⁻⁴m [¹⁴C]tyramine in 0.17 m sodium acetate at pH 4.0–6.0. Activities were determined from the formation of [¹⁴C]octopamine, after periodate oxidation and extraction, measured by using liquid scintillation counting. Regardless of species, maximal activities could be obtained at low substrate concentrations only by adding 3.5 to 10 μm copper to the assay. Following the addition of NEM, DBH activities were suboptimal. Incremental NEM additions to a spectrophotometric assay at saturating tyramine concentrations resulted in maximal activity only in human plasma. With optimal copper in the radioenzymatic assay, representative human values ranged from 2.1 ± 0.2 to 7.4 ± 0.4 nmole/min/ml of plasma (mean ± SEM) at pH 5.5. Rat, rabbit, and dog ranged 100-fold less in activity and varied with respect to pH optimum. Studies with purified internal standards defined the influence of copper and NEM on endogenous inhibitor neutralization. Our results support the proposal that precise determinations of plasma DBH activities are limited by conditions within the assay.     

Light- and electron-microscopic studies on the formation of “dark” variants of chromaffin cells in the rat adrenal medulla

Summary Dark cell formation has been studies in the adrenal medulla with combined light- and electron-microscopic methods. Correlation between light- and electron-microscopic appearance has been demonstrated. It has been pointed out that the formation of dark cell artefact can be prevented during immersion fixation and can be produced also during perfusion fixation by appropriate physical conditions.Dark cell formation was elicited by prefixation cooling and/or by increased tissular pressure. The nature of the underlying physicochemical change was interpreted as a uniformly distributed sol-gel transition of cytoplasmic macromolecules. The formation of cytoplasmic gel structures can be considered an endothermic process which involves volume increase (V)-as may be inferred by their susceptibility to solation by high pressure and low temperature.     

Simultaneous evaluation of mRNAs of dopamine β-hydroxylase,tyrosine hydroxylase and proenkephalin a from three human pheochromocytomas

Using the rabbit reticulocyte cell-free translation system, the relative proportions of in vitro translatable mRNAs of three proteins in three human pheochromocytomas: tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and proenkephalin A have been compared.TH expression appeared rather constant in the three tumors. In contrast, those of DBH and proenkephalin A were more variable. Though the actual level of each mRNA was not determined, the identical value of DBH/proenkephalin A mRNAs ratio in the three tumors could suggest a coordination in the expression of these two proteins.     

P25α / TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity

Parkinson's disease is characterized by preferential degeneration of the dopamine-producing neurons of the brain stem substantia nigra. Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25α accumulates in dopaminergic neurons in Parkinson's disease. We hypothesized that p25α modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity. Here we show that co-expression of the dopamine transporter with p25α in HEK-293-MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter. No direct interaction between p25α and the dopamine transporter was demonstrated, but they co-fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25α peptides to brain vesicles was demonstrated. Truncations of the p25α peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding. Co-expression of p25α and the dopamine transporter in HEK-293-MSR cells sensitized them to the toxicity of extracellular dopamine. Neuronal expression of p25α thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter.