Regulation Of Catecholamine Secretion Research Articles

Phorbol Esters Cause Preferential Secretion of Norepinephrine from Bovine Chromafin Cells

Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13-dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine-containing cells or epinephrine-containing cells. In response to 55 mM K+, catecholamine release from norepinephrine-containing cells was two times greater than that from epinephrine-containing cells. In response to phorbol 12,13-dibutyrate, secretion from norepinephrine-containing cells was 13 times greater than that from epinephrine-containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine-containing cells.

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.

Substance P increases catecholamine secretion from perfused rat adrenal glands evoked by prolonged field stimulation.

1. We have studied the effect of substance P (SP) on catecholamine (CA) secretion evoked by prolonged field stimulation from the perfused rat adrenal gland in vitro. 2. Previous studies show that SP has an inhibitory effect on the nicotinic response in a number of different tissue preparations. In the present study, we found that SP at concentrations from 10(-7) to 10(-6) M markedly increased CA secretion evoked by prolonged high-frequency field stimulation but not that evoked by low-frequency field stimulation. 3. In the absence of field stimulation SP by itself had no direct effect on CA secretion. This indicates that SP acts as a neuromodulator rather than a neurotransmitter to increase CA secretion from the adrenal medulla. 4. The nicotinic receptors were still functional following a period of prolonged field stimulation (120 min) because CA secretion was markedly increased in response to the nicotinic agonist dimethylphenylpiperazinium (DMPP). 5. Capsaicin pre-treatment reduced CA secretion evoked by prolonged field stimulation and the facilitatory action of SP on CA secretion lasted longer in these capsaicin-pre-treated rats than in controls, indicating that SP-containing capsaicin-sensitive fibres innervating the adrenal medulla may be involved in the regulation of CA secretion. 6. In parallel with the increase in CA secretion, 3H overflow from the splanchnic nerve pre-labelled with [3H]choline was also increased by SP. The increase in CA secretion by SP lasted longer than the increase in 3H overflow. These results suggest that SP facilitates CA secretion from the adrenal gland at two levels: (1) pre-synaptically by facilitating ACh release from splanchnic nerve terminals, and (2) post-synaptically by modulating the nicotinic secretory response by protection against nicotinic desensitization of secretion. 7. The present studies provide further evidence that endogenous SP in the splanchnic nerve may modulate CA secretion during stress.

Binding of prostaglandin E 2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E 1 and E 2. We have now investigated the binding of PGE 2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE 2 with a dissociation constant of 2 nM and a concentration of about 40 000 binding sites per cell. Low concentrations of PGE 2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE 2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 μM) of PGE 2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE 2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE 2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.

Secretion from chromaffin cells is controlled by chromogranin A-derived peptides.

Chromogranin A (CGA) is the major protein of the secretory granule from chromaffin cells and also is found in a variety of endocrine cells. Although the sequence of this acidic glycoprotein has been elucidated recently, its biological function is unknown. Here we have purified CGA from chromaffin granules; the final preparation contained the 74-kDa native CGA together with two degradation products--three bands near 60 kDa and a single band of 43 kDa. This preparation was found to inhibit (a maximum inhibition of 60% at 1 microM) the nicotine-induced, but not the high K+-evoked, catecholamine secretion from bovine chromaffin cells maintained in primary culture. Spontaneous release was also affected in the nanomolar CGA protein concentration range. The observation that the inhibitory effect is strictly dependent on a preincubation step together with the modification of the CGA protein profile during this preincubation step suggests that the degradation peptide(s) rather than the 74-kDa native CGA--the approximately equal to 60-kDa bands or the 43-kDa singlet band--is actually involved in secretory cell activity. This was demonstrated by using trypsin-generated peptides that were inhibitory without the preincubation period. The finding that unprocessed CGA is not active on chromaffin cell secretion suggests that this molecule is a precursor of a peptide(s) that is able to regulate catecholamine secretion. Thus, the present data suggest that a CGA-derived peptide(s) could exert a feedback control on chromaffin cell secretory activity--a mechanism that might be of importance during stress situations.

Influence of reduction of cytoplasmic ATP on catecholamine secretion from intact and digitonin-permeabilized adrenal chromaffin cells.

The effect of the metabolic inhibitor 2,4-dinitrophenol (DNP) on the concentration of cytoplasmic ATP and the activity of catecholamine secretion was studied using cultured bovine adrenal chromaffin cells. The pretreatment of the cells with DNP resulted in a decrease in the concentration of cytoplasmic ATP. Catecholamine secretion evoked by either carbamylcholine or high K+ was also reduced by DNP pretreatment. Oligomycin caused a significant decrease in both cytoplasmic ATP concentration and catecholamine secretion. In contrast, neither the ATP level nor the secretory response was affected by 2-deoxyglucose. Similarly, catecholamine release from digitonin-permeabilized chromaffin cells in response to a direct calcium challenge was reduced by pretreatment of the cells with DNP, and this reduction was partially prevented by the presence of ATP in the permeabilizing medium. These results suggest that ATP in the cell cytoplasm may play an important role in the regulation of catecholamine secretion as a factor modulating the activity of the exocytotic process in the adrenal chromaffin cell.

Influence of Reduction of Cytoplasmic ATP on Catecholamine Secretion from Intact and Digitonin-Permeabilized Adrenal Chromaffin Cells

The effect of the metabolic inhibitor 2,4-dinitrophenol (DNP) on the concentration of cytoplasmic ATP and the activity of catecholamine secretion was studied using cultured bovine adrenal chromaffin cells. The pretreatment of the cells with DNP resulted in a decrease in the concentration of cytoplasmic ATP. Catecholamine secretion evoked by either carbamylcholine or high K+ was also reduced by DNP pretreatment. Oligomycin caused a significant decrease in both cytoplasmic ATP concentration and catecholamine secretion. In contrast, neither the ATP level nor the secretory response was affected by 2-deoxyglucose. Similarly, catecholamine release from digitonin-permeabilized chromaffin cells in response to a direct calcium challenge was reduced by pretreatment of the cells with DNP, and this reduction was partially prevented by the presence of ATP in the perme-abilizing medium. These results suggest that ATP in the cell cytoplasm may play an important role in the regulation of catecholamine secretion as a factor modulating the activity of the exocytotic process in the adrenal chromaffin cell.

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 ? M) inhibited CA release evoked by acetylcholine (ACh), excess K(+) and veratridine but not by A23187 or caffeine in Ca(2+) -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-?-carboline-3-carboxylate (?CCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K(+) was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked (45)Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca(2+) influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.

Neonatal adaptation: naloxone increases the catecholamine surge at birth.

A marked increase in plasma catecholamines at birth has been described in animals and man. Because the factors that regulate catecholamine secretion are incompletely understood and because it has recently been suggested that endogenous opiates are important in the regulation of catecholamine secretion, we designed studies to determine the influence of opiate receptor blockade prior to delivery on the increase in plasma catecholamines at birth. Term fetal sheep were delivered by cesarean section and randomly assigned to receive naloxone or vehicle. Naloxone was given just prior to umbilical cord cutting as a 2 mg/kg bolus followed by 2 mg/kg/h. Naloxone administration resulted in significantly greater peak levels of plasma norepinephrine (peak levels of 1.5 +/- 0.4 versus 0.9 +/- 0.1 ng/ml) and epinephrine (peak levels of 1.4 +/- 0.7 versus 0.9 +/- 0.3 ng/ml) and higher norepinephrine values throughout the study period. Naloxone administration was associated with significantly elevated heart rate (peak 184 +/- 12 versus 207 +/- 13 beats per min) and blood pressure (peak 95 +/- 6 versus 88 +/- 2 mm Hg). These studies demonstrate that opiate receptor blockade from birth markedly augments the neonatal sympathoadrenal response in the term newborn lamb.

Studies on adrenorphin in pheochromocytoma.

We studied the secretion and tissue contents of adrenorphin in human pheochromocytomas. In 17 human pheochromocytomas from 11 patients, we found a remarkably wide distribution in immunoreactive adrenorphin levels (3-7771 pg/mg tissue). Adrenomedullary pheochromocytomas contained a significantly larger amount of immunoreactive adrenorphin (2295 +/- 1092 pg/mg, mean +/- SE) than did extramedullary ones (17.8 +/- 8.4 pg/mg). Gel chromatographic studies revealed that immunoreactive adrenorphin consisted largely of material emerging at the position of synthetic adrenorphin in both pheochromocytoma and normal adrenal medulla tissue. Nicotine (10(-5) M) significantly stimulated the secretion of immunoreactive adrenorphin as well as catecholamines from cultured human pheochromocytoma cells. Adrenorphin was a more potent inhibitor of catecholamine secretion evoked by 10(-5) M nicotine than was met-enkephalin in cultured human pheochromocytoma cells. The 50% inhibitory concentrations (IC50) were 1.1 X 10(-6) and 6.5 X 10(-5) M for adrenorphin and met-enkephalin, respectively. The effect of adrenorphin was much the same as that of dynorphin-(1-13) (IC50, 1.0 X 10(-6) M) and BAM-12P (IC50, 4.5 X 10(-6) M). These results indicate the presence and secretion of adrenorphin in human pheochromocytomas. Adrenorphin may play an important role in regulating catecholamine secretion in human pheochromocytoma.

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators.

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 cells.

Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium.

Although the mechanism by which nicotinic receptors on adrenal chromaffin cells regulate catecholamine secretion is reasonably well understood, that of the muscarinic receptors remains obscure. The effects of both acetylcholine and specific muscarinic agonists on cytosolic free calcium in isolated bovine adrenal chromaffin cells have been measured using the fluorescent probe Quin-2. Acetylcholine (0.1 mM) evokes a large increase in cytosolic free calcium from resting levels near 100 nM into the microM range, most of which is blocked by hexamethonium (0.5 mM) or removal of extracellular calcium. A small component of the acetylcholine-evoked rise in cytosolic free calcium (approximately 50-100 nM) is independent of extracellular calcium and is unaffected by 0.5 mM hexamethonium, but is totally blocked by 0.5 microM atropine. The muscarinic nature of this component is further confirmed by the fact that the muscarinic agonists, muscarine (0.1 mM) and methacholine (0.3 mM), stimulate a 50-100 nM rise in chromaffin cell cytosolic calcium which is blocked by 0.5 microM atropine and is largely independent of extracellular calcium. These results suggest that muscarinic receptors regulate cytosolic calcium in chromaffin cells by a new mechanism different from that of nicotinic receptors, a mechanism utilizing an intracellular calcium source. The small size of the muscarinic-induced rise in cytosolic calcium in the bovine chromaffin cell would explain why no secretion is evoked by muscarinic agonists in this species.

Choline administration: Lack of effect on plasma catecholamines in rats

Choline chloride (35 or 70 mg/kg, IP) or saline was administered daily for 3 consecutive days to adult male Sprague-Dawley rats. Before and 30, 60 and 120 minutes after the third injection of choline chloride or saline, blood samples were collected from a chronic tail artery catheter and later analyzed for levels of norepinephrine (NE) and epinephrine (EPI). Plasma levels of both catecholamines did not differ between choline- and saline-injected rats at either of the four sampling points. When insulin (10 IU/kg, SC) was administered to stimulated the sympathetic-adrenal medullary system reflexy, plasma levels of NE and EPI increased significantly above basal values but were similar for choline- and saline-injected rats. These findings do not support a role for choline availability in the regulation of catecholamine secretion from the adrenal medulla.

Somatostatin and substance P inhibit catecholamine secretion from isolated cells of guinea-pig adrenal medulla

Chromaffin cells isolated from guinea-pig adrenal glands secrete catecholamines in response to acetylcholine, nicotine, pilocarpine, veratridine, and high [K +]. Both substance P and somatostatin inhibit acetylcholine-induced catecholamine secretion. The maximal inhibition of acetylcholine-induced catecholamine secretion produced by substance P and by somatostatin is approximately 60%: the concentrations of the peptides required for half-maximal inhibition of secretion are approximately 0.8 and 2 μ M. respectively. The maximal inhibition of catecholamine secretion produced by somatostatin and that caused by substance P are not additive. The effects of the peptides on secretion are readily reversible. Somatostatin and substance P also inhibit nicotine-induced catecholamine secretion, but they do not inhibit catecholamine secretion stimulated by pilocarpine, veratridine, or high [K +]. Thus, these peptides specifically inhibit catecholamine secretion linked to stimulation of nicotinic receptors. The inhibition of acetylcholine-induced catecholamine secretion by somatostatin is noncompetitive with respect to acetylcholine, Na + or Ca 2+. Immunoreactive somatostatin and substance P are present in guinea-pig adrenal glands. It is suggested that these peptides may play a role in the regulation of catecholamine secretion from the adrenal medulla.