Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.     

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg²⁺ and Zn,²⁺ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca²⁺ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Peroxisome proliferators as inducers and substrates of UDP-glucuronosyltransferases

Summary— Peroxisome proliferators, despite their chemically unrelated structures, share the common property of being able to stimulate the glucuronidation of bilirubin in rodents and, probably, also in man. The aryloxycarboxylic acids (clofibric acid, fenofibrate, bezafibrate, ciprofibrate), tiadenol and probucol, all of which have hypolipidemic properties, as well as the fatty acid-like perfluorodecanoic acid all enhanced the expression of the UDP-glucuronosyltransferase (UGT) form involved in the conjugation of the pigment. This induction is manifested by an increase in the mRNA species encoding the protein with a subsequent increase in the neosynthesis of the corresponding protein in the endoplasmic reticulum. The induction process is concomitant with that of cytochrome P-450-IVA1 and cytosolic epoxide hydrolase, which, like bilirubin UGT, are mainly involved in the metabolism of endogenous substrates. With a series of carboxylic acids related to clofibric acid, it was possible to demonstrate that induction was mediated via specific interactions based on the physicochemical properties of the inducers. Until now, the molecular basis of induction of bilirubin UGT is not known. The peroxisome proliferators that possess a carboxyl group are good substrates of UGT, especially in man. The acylglucuronides formed are known for their instability and reactivity which could contribute to the toxicity encountered in some patients treated with the drugs. There is convincing evidence that UGT bilirubin does not catalyze the glucuronidation of these substances even if the two types of substrate form acylglucuronides.     

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.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Tyrosine hydroxylase in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.