Action potentials in the rat chromaffin cell and effects of acetylcholine.
1. Electrophysiological properties of the rat chromaffin cell were studied using intracellular recording techniques. 2. The resting potential in the chromaffin cell was -49 +/- 6 mV (mean +/- S.D., n = 14) in standard saline containing 10 mM-Ca whereas that in Na-free saline was -63 +/- 9 mV (n = 17). At rest, the membrane has a substantial Na permeability. 3. Action potentials were evoked by passing current through the recording electrode. In standard saline the major fraction of the action potential disappeared either upon omission of external Na ions from standard saline or addition of 1 muM tetrodotoxin (TTX). We conclude that action potentials in the chromaffin cell are due mainly to an increase in the permeability of the membrane to Na ions. 4. Small but significant regenerative action potentials were observed in Na-free saline, and when Ca in Na-free saline was replaced by Ba, prolonged action potentials occurred. We conclude that action potentials in the chromaffin cell also have a Ca component. 5. Iontophoretic application of acetylcholine (ACh) produced a transient membrane depolarization in standard saline. 6. Spontaneous action potentials were recorded extracellularly by microsuction electrodes. They occurred at a rate of 0-05-0-1/sec in almost all cells. 7. When the perfusion fluid contained 3 x 10(-7) M to 10(-4) M ACh the spike frequency increased up to about 2/sec. This stimulatory effect of ACh was blocked by 10(-7) M atropine but not by 10(-3) M hexamethonium nor by 10(-5) M-d-tubocurarine. 8. The importance of Ca entry during action potentials for catecholamine secretion is discussed (+info)
Modulation of gastrin processing by vesicular monoamine transporter type 1 (VMAT1) in rat gastrin cells.
1. Gastrointestinal endocrine cells produce biogenic amines which are transported into secretory vesicles by one of two proton-amine exchangers, vesicular monoamine transporters type 1 and 2 (VMAT1 and 2). We report here the presence of VMAT1 in rat gastrin (G) cells and the relevance of VMAT1 function for the modulation of progastrin processing by biogenic and dietary amines. 2. In immunocytochemical studies VMAT1, but not VMAT2, was localized to subpopulations of G cells and enterochromaffin (EC) cells; neither was found in antral D cells. The expression of VMAT1 in antral mucosa was confirmed by Northern blot analysis, which revealed an mRNA band of approximately 3.2 kb, and by Western blot analysis, which revealed a major protein of 55 kDa. 3. In pulse-chase labelling experiments, the conversion of the amidated gastrin G34 to G17 was inhibited by biogenic amine precursors (L-DOPA and 5-hydroxytryptophan). This inhibition was stereospecific and sensitive to reserpine (50 nM), which blocks VMAT1 and VMAT2, but resistant to tetrabenazine, which is a selective inhibitor of VMAT2. 4. Dietary amines such as tyramine and tryptamine also inhibited G34 cleavage. This effect was associated with a loss of the electron-dense core of G cell secretory vesicles. It was not stereospecific or reserpine sensitive, but was correlated with hydrophobicity. 5. Thus rat antral G cells can express VMAT1; transport of biogenic amines into secretory vesicles by VMAT1 is associated with inhibition of G34 cleavage, perhaps by raising intravesicular pH. Dietary amines also modulate cleavage of progastrin-derived peptides, but do so by a VMAT1-independent mechanism; they may act as weak bases that passively permeate secretory vesicle membranes and raise intravesicular pH. (+info)
Subcellualr distribution of protein carboxymethylase and its endogenous substrates in the adrenal medulla: possible role in excitation-secretion coupling.
Protein carboxymethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 18.104.22.168) transfers a methyl group from S-adenoxyl-L-methionine to carboxyl side chains of proteins to form labile protein-methyl esters which, thus, neutralize negative charges. This enzyme was examined for its possible participation in excitation-secretion coupling in the adrenal medulla. Protein carboxymethylase has a specific activity several times higher in the adrenal medulla than in the adrenal cortex; also, the medulla has a higher concentration of methyl-acceptor proteins. In the adrenal medulla, 97% of the enzyme was localized in the cytosol. Of the various subcellular fractions of the medulla, the catecholamine-containing chromaffin vesicles had the highest concentrations of substrat(s) for protein carboxymethylase. Carboxymethylation of proteins in intact chromaffin vesicles results in stripping of methylated protein(s) from the membranes. Thus, protein carboxymethylase appears to be involved in the neutralization of charges on the surface of chromaffin vesicles and in the release of surface proteins; both phenomena are likely to be required for exocytosis. (+info)
Desensitisation of chromaffin cell nicotinic receptors does not impede catecholamine secretion during acute hypoxia in rainbow trout (Oncorhynchus mykiss).
Experiments were performed on adult rainbow trout (Oncorhynchus mykiss) in vivo using chronically cannulated fish and in situ using a perfused posterior cardinal vein preparation (i) to characterise the desensitisation of chromaffin cell nicotinic receptors and (ii) to assess the ability of fish to secrete catecholamines during acute hypoxia with or without functional nicotinic receptors. Intra-arterial injection of nicotine (6.0x10(-)(7 )mol kg(-)(1)) caused a rapid increase in plasma adrenaline and noradrenaline levels; the magnitude of this response was unaffected by an injection of nicotine given 60 min earlier. Evidence for nicotinic receptor desensitisation, however, was provided during continuous intravenous infusion of nicotine (1.3x10(-)(5 )mol kg(-)(1 )h(-)(1)) in which plasma catecholamine levels increased initially but then returned to baseline levels. To ensure that the decline in circulating catecholamine concentrations during continuous nicotine infusion was not related to changes in storage levels or altered rates of degradation/clearance, in situ posterior cardinal vein preparations were derived from fish previously experiencing 60 min of saline or nicotine infusion. Confirmation of nicotinic receptor desensitisation was provided by demonstrating that the preparations derived from nicotine-infused fish were unresponsive to nicotine (10(-)(5 )mol l(-)(1)), yet remained responsive to angiotensin II (500 pmol kg(-)(1)). The in situ experiments demonstrated that desensitisation of the nicotinic receptor occurred within 5 min of receptor stimulation and that resensitisation was established 40 min later. The ability to elevate plasma catecholamine levels during acute hypoxia (40-45 mmHg; 5.3-6.0 kPa) was not impaired in fish experiencing nicotinic receptor desensitisation. Indeed, peak plasma adrenaline levels were significantly higher in the desensitised fish during hypoxia than in controls (263+/-86 versus 69+/-26 nmol l(-)(1); means +/- s.e.m., N=6-9). Thus, the results of the present study demonstrate that activation of preganglionic sympathetic cholinergic nerve fibres and the resultant stimulation of nicotinic receptors is not the sole mechanism for eliciting catecholamine secretion during hypoxia. (+info)
Tumours of the adrenal gland and paraganglia.
This classification is arranged in two parts in order to take into account the different origins, structures, and functions of the cortex and medulla. The tabular classification is a simplified version of that suggested for adrenal tumours in man, and includes cortical adenoma and carcinoma, phaeochromocytoma, chemodectoma, neurofibroma, ganglioneuroma and ganglioneuroblastoma, and neuroblastoma. A detailed functional classification is not given, since the hormonal activity of many adrenal tumours in animals is less well known than it is in man. Of the tumour-like lesions listed, cortical hyperplasia is particularly important in several species. (+info)
Ion permeability of isolated chromaffin granules.
The passive ion permeability, regulation of volume, and internal pH of isolated bovine chromaffin granules were studied by radiochemical, potentiometric, gravimetric, and spectrophotometric techniques. Chromaffin granules behave as perfect osmometers between 340 and 1,000 mosM in choline chloride, NaCl, and KCl as measured by changes in absorbance at 430 nm or from intragranular water measurements using 3H2O and [14C]polydextran. By suspending chromaffin granules in iso-osmotic media of various metal ions and selectively increasing the permeability to either the cation or the anion by intrinsically permeable ions or specific ionophores, it was possible to determine by turbidity and potentiometric measurements the permeability to the counterion. These measurements indicate that the chromaffin granule is impermeable to the cations tested (Na+, K+, and H+). Limited H+ permeability across the chromaffin granule membrane was also shown by means of the time course of pH re-equilibration after pulsed pH changes in the surrounding media. The measurement of [14C]methylamine distribution indicates that a significant deltapH exists across the membrane, inside acidic, which at an external value of 6.85 has a value of 1.16. The deltapH is relatively insensitive to changes in the composition of the external media and can be enhanced or collapsed by the addition of ionophores and uncouplers. Measurement at various values of external pH indicates an internal pH of 5.5. Use of the ionophore A23187 indicates that Ca++ and Mg++ can be accumulated against an apparent concentration gradient with calcium uptake exceeding 50 nmol/mg of protein at saturation. These measurements also show that Ca++ and Mg++ are impermeable. Measurement of catecholamine release under conditions where intravesicular calcium accumulation is maximal indicates that catecholamine release does not occur. The physiological significance of the high impermeability to ions and the existence of a large deltapH are discussed in terms of regulation of uptake, storage, and release of catecholamines in chromaffin granules. (+info)
Release of catecholamines and dopamine beta-hydroxylase from the perfused adrenal gland of the cat.
1. Secretion of catecholamines (CA) and dopamine beta-hydroxylase (DBH) activity from the perfused cat adrenal gland was studied following splanchnic nerve stimulation or infusion of acetylcholine (ACh). 2. Splanchnic nerve stimulation (30 Hz) or perfusion with a low concentration of ACh (10-minus5 M) caused a marked release of CA in the venous effluent, but release of DBH activity was minimal while a higher concentration of ACh (10-minus 4 M) enhanced the release of CA and DBH. 3. The ratio of DBH/CA released in the perfusate by splanchnic nerve stimulation or ACh infusion was only a small fraction of the ratio in the soluble lysate of purified chromaffin vesicles. 4. Following reserpine treatment, adrenal CA levels fell to 25% of the control value in 24 hr, remained depressed on days 2, 3, 4 and 5 at 5% of the control and recovered to 60% of the control value on the 6th day. DBH activity was unchanged from the control value at 24 hr after treatment, then rose as high as 5 times the control on the 5th day and was still twice the control value on the 6th day. 5. CA secretion in response to ACh (10-minus 4 M) perfusion was reduced to 30% of the control value on the first day after reserpine treatment, while DBH secretion was unchanged. On the 2nd day, CA secretion was depressed further to 5% of the control and remained at this low level up to 5 days after treatment while DBH secretion was twice the control value at 48 hr and then on days 3, 4 and 5 rose up to 5 times the control value. On the 6th day, secretion of CA recovered to 30% of the control while DBH secretion was now twice the control. 6. Isopycnic sucrose density (discontinuous) gradient centrifugation of vesicles from adrenal glands of control cats, and of cats given reserpine 1 or 2 days perviously, indicated that new vesicles or vesicles depleted of CA by reserpine had a lower equilibrium density than the original population of vesicles. 7. These results suggest that the release of CA is quantal in nature, but the release of DBH is not necessarily coupled with it. Release of DBH by ACh from reserpinized glands suggests that the vesicles which were once involved in secretion may be re-used for synthesis and storage of CA. (+info)
Discrimination of monoamine uptake by membranes of adrenal chromaffin granules.
1 The accumulation of various radioactive monoamines by isolated membranes of bovine adrenal chromaffin granules was measured by equilibrium dialysis. 2 Adenosine-5'-triphosphate (ATP) in the presence of Mg++ stimulated the uptake of all the amines tested, but the accumulation of dopamine, (-)-noradrenaline (NA), 5-hydroxytryptamine (5-HT), (plus or minus)-adrenaline and (plus or minus)-octopamine was greater than that of tyramine, (plus or minus)-metaraminol, tryptamine, beta-phenylethylamine and histamine. 3 At the higher concentration levels of the amines in the medium the ATP-dependent accumulation of dopamine, NA, adrenaline and 5-HT in the membranes reached a saturation level, whereas in the absence of the nucleotide no saturation level was attained. 4 Octopamine and 5-HT competitively inhibited the ATP-dependent uptake of NA, 5 Decrease in the incubation temperature or the presence of N-ethylameimide greatly reduced the ATP-stimulated amine accumulation. Ouabain had no effect on uptake. 6 Reserpine virtually abolished the ATP-dependent uptake of dopamine, NA and 5-HT, caused a partial inhibition of the metaraminol, octopamine and tyramine accumulation, but did not interfere with the uptake of tryptamine. 7 The content of endogenous catecholamines of the membranes was changed very little by incubation of NA and 5-HT in the presence of ATP. However, the membranes lost over 80% of their endogenous amines if incubated for 30 min without ATP. 8 The ATP content of the medium progressively decreased during the incubation of granular membranes. 9 It is concluded that the membrane of adrenal chromaffin granules discriminates between the various monoamines with regard to the magnitude of their uptake and that two mechanisms of ATP-stimulated uptake, one responsive and the other resistant to reserpine, exist at the level of this membrane. The ATP-stimulated transport at the granular membrane level may be an important factor in determining the intraneuronal storage of a physiological or false neurotransmitter. (+info)