B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Vatta, M. S., M. F. Presas, L. G. Bianciotti, M. Rodriguez–fermepin, R. Ambros and B. E. Fernandez. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of ³H-NE. Experiments were carried out in rat adrenal medulla slices incubated “in vitro.” Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.     

Atrial natriuretic factor inhibits noradrenaline release in the presence of angiotensin II and III in the rat hypothalamus

1. Atrial natriuretic factor effects on neuronal noradrenaline release evoked by angiotensin II or III and high potassium solution plus angiotensin II and III in the rat hypothalamus were studied.2. Atrial natriuretic factor (10 nM) did not modify spontaneous noradrenaline release. On the other hand, the atrial factor diminished the increase of noradrenaline release induced by both angiotensin II (1 μM) or angiotensin III (1 μM).3. Ten nanomolar ANF reduced the amine output induced by 100 nM KCl. Both angiotensins enhanced the 3H-noradrenaline secretion stimulated by high potassium solutions. When atrial natriuretic factor was added to the medium containing the depolarizing KCl solution plus angiotensin II or III (1 μM), the diminishing effects were greater than when the atrial factor was added to the depolarizing solution alone.4. Our results suggest that atrial natriuretic factor effects on noradrenaline release, evoked by angiotensin II, III and KCl, may be involved in the regulation of the central catecholamine pathways and sympathetic activity.     

Effect of atrial natriuretic peptide on acetylcholine-induced release of corticotropin-releasing factor from rat hypothalamus in vitro

Effect of rat atrial natriuretic peptide (rANP) on acetylcholine-induced release of corticotropin-releasing factor (CRF) from the rat hypothalamus was studied in vitro using perifusion method. Perifused acetylcholine at 100 and 1000 ng/ml evoked significant CRF release, whereas norepinephrine at 10, 100 and 1000 ng/ml did not show a definite effect on CRF release. Continuous administration of alpha-rANP(1-28) (20ng/ml) inhibited the acetylcholine (100ng/ml)-induced CRF release. It is likely that ANP is involved in the regulation of CRF release.     

Role of endogenous PACAP in catecholamine secretion from the rat adrenal gland

We elucidated the contribution of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) to neurally evoked catecholamine secretion from the isolated perfused rat adrenal gland. Infusion of PACAP (100 nM) increased adrenal epinephrine and norepinephrine output. The PACAP-induced catecholamine output responses were inhibited by the PACAP type I receptor antagonist PACAP- (6-38) (30-3,000 nM) but were resistant to the PACAP type II receptor antagonist [Lys1,Pro2,5,Ara3,4,Tyr6]-vasoactive intestinal peptide (LPAT-VIP; 30-3,000 nM). Transmural electrical stimulation (ES; 1-10 Hz) or infusion of ACh (6-200 nM) increased adrenal epinephrine and norepinephrine output. PACAP-(6-38) (3,000 nM), but not LPAT-VIP, also inhibited the ES-induced catecholamine output responses. However, PACAP-(6-38) did not affect the ACh-induced catecholamine output responses. PACAP at low concentrations (0.3-3 nM), which had no influence on catecholamine output, enhanced the ACh-induced catecholamine output responses, but not the ES-induced catecholamine output responses. These results suggest that PACAP is released from the nerve endings to facilitate the neurally evoked catecholamine secretion through PACAP type I receptors in the rat adrenal gland.     

Effects of atrial natriuretic peptide and angiotensin III on the uptake and intracellular distribution of norepinephrine in medulla oblongata of the rat.

1. Ten micromoles angiotensin III decreased total 3H-norepinephrine uptake in medulla oblongata of the rat and 100 nM atrial natriuretic peptide increased it. These were the threshold concentrations for the peptides to modify the uptake of the amine. 2. A threshold concentrations (1 nM) of atrial natriuretic peptide reversed the effects produced by 10 microM angiotensin III on total 3H-norepinephrine uptake, but subthreshold angiotensin III concentrations failed to alter the effects produced by 100 nM atrial natriuretic peptide. 3. Angiotensin III, as well as atrial natriuretic peptide, modified only neuronal norepinephrine uptake and did not alter non-neuronal norepinephrine uptake. 4. Angiotensin III and atrial natriuretic peptide did not modify the intracellular distribution of norepinephrine in medulla oblongata.     

Role of potassium channels in catecholamine secretion in the rat adrenal gland

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.     

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.     

Tumor Necrosis Factor Modulates the Inactivation of Catecholamine Secretion in Cultured Sympathetic Neurons

Cytokines exert multiple effects on cellular functions. We studied the effects of cytokines on the calcium-dependent release of catecholamines in cultured neurons from neonatal rat superior cervical ganglia. Incubation of sympathetic neurons with recombinant human interleukin-1 beta (0.14-0.7 nM) or recombinant human tumor necrosis factor-alpha (1 nM) for 24-48 h had no effect on the baseline spontaneous release and the initial K(+)-evoked [3H]norepinephrine release, compared with untreated cells. A repeat K(+)-induced depolarization after 6 min resulted in a decrease of [3H]norepinephrine secretion to 69 +/- 5.8% (n = 11) of the initial secretion in recombinant human tumor necrosis factor-treated cells, but not in control cells. The secretory response was restored when the interval between the two K+ challenges was increased to 10 min. We conclude that the diminished secretory response to a repeat stimulus in recombinant human tumor necrosis factor-treated superior cervical ganglia neurons is due to a prolonged recovery from inactivation of secretion in these cells.     

Stimulatory effect of acetylcholine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of acetylcholine (Ach) and gamma-aminobutyric acid (GABA) on immunoreactive corticotropin-releasing factor (CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Ach stimulated CRF release in a dose-dependent manner (1 pM-1 nM). One nM Ach-induced CRF release was inhibited by atropine in a dose-dependent manner (1-100 nM), but was inhibited by only a high concentration (100 nM) of hexamethonium. In addition, such Ach-induced CRF release was inhibited by norepinephrine. GABA did not influence basal CRF release. These results suggest that Ach stimulates CRF release mainly through muscarinic receptors at least under our conditions.     

The release of 3H-1-methyl-4-phenylpyridinium from bovine adrenal chromaffin cells is modulated by somatostatin

Besides cholinergic regulation, catecholamine secretion from adrenal chromaffin cells can be elicited and/or modulated by noncholinergic neurotransmitters and hormones. This study was undertaken to investigate the influence of somatostatin and octreotide on [3H]MPP+ secretion evoked by KCl or cholinergic agents, from bovine adrenal chromaffin cells. The release of [3H]MPP+ was markedly increased by excess KCl (50 mM), acetylcholine (50 microM-10 mM) and by the nicotinic agonists, nicotine (5-100 microM) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10-100 microM), but not by the muscarinic agonist, pilocarpine (10-100 microM). Acetylcholine-evoked release of [3H]MPP+ from these cells was mainly mediated by nicotinic receptors: a) nicotine and DMPP stimulated the release of [3H]MPP+, b) a nicotinic antagonist, hexamethonium, markedly blocked the acetylcholine-evoked response and c) pilocarpine was devoid of effect on [3H]MPP+ secretion. At all concentrations tested, somatostatin and octreotide interfered neither with [3H]MPP+ basal release nor with KCl-induced release of [3H]MPP+. However, somatostatin (0.01-0.3 microM) increased the release of [3H]MPP+ induced by a high concentration of acetylcholine (10 mM). Octreotide (1-10 microM) had no effect. These results, showing that somatostatin potentiates acetylcholine-induced [3H]MPP+ release, support the hypothesis that somatostatin may increase the release of catecholamines from adrenal medullary cells.     

Atrial natriuretic factor increases the magnitude of duodenal spontaneous phasic contractions

The present investigation was designed to determine if atrial natriuretic factor relaxes non-vascular smooth muscle. Rather than cause a relaxation, atrial natriuretic factor induced a two-to-four fold enhancement in the amplitude of the spontaneous phasic contractions of duodenal longitudinal muscle. Dose-response curves revealed that ANF enhanced these contractions over a concentration range of 10 picomoles to 100 nanomoles with the ED50 at 1 nanomolar. The increased amplitude of contraction began within 30 seconds and was calcium-dependent. The increased force of contraction was associated with a three-fold increase in cyclic GMP levels and activation of particulate guanylate cyclase [E.C.4.5.1.2.]. Atrial natriuretic factor had its half-maximal [ED50] activation of guanylate cyclase at its 1 nM concentration while maximal enhancement was at its 100 nM concentration in duodenum, jejunum, and ileum. Atrial natriuretic factor did not stimulate adenylate cyclase [E.C.4.6.1.1.]. Thus, atrial natriuretic factor increases the force of the spontaneous phasic contractions of the small intestine which are calcium-dependent and associated with activation of the guanylate cyclase-cyclic GMP system.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Novel 1,4-Dihydropyridine (Bay K 8644) Facilitates Calcium-Dependent [3H]Noradrenaline Release from PC 12 Cells

The effects of the novel 1,4-dihydropyridine Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate] on the release of [3H]noradrenaline in cultured PC 12 cells were investigated. K+ in a concentration-dependent manner evoked 3H-transmitter release with an EC50 of 50-56 mM. Bay K 8644 at 30 nM potentiated the K+-evoked [3H]noradrenaline release; however, in the absence of calcium neither K+ evoked nor Bay K 8644 enhanced [3H]noradrenaline release. At a K+ concentration of 25 mM, Bay K 8644 stimulated [3H]noradrenaline release fivefold, with an EC50 of 10 nM, and 100 nM of the calcium channel blocker nitrendipine shifted the concentration response curve of Bay K 8644 to the right in an apparently competitive fashion. Nitrendipine blocked the Bay K 8644-potentiated release with an EC50 of 700 nM in the presence of 500 nM Bay K 8644. [3H]Nitrendipine bound to a saturable population of binding sites on PC 12 cell membranes with a Bmax of 180 fmol X mg-1 of membrane protein and a KD of 0.9 nM. Bay K 8644 inhibited [3H]nitrendipine binding with a Ki of 16 nM. It is concluded that Bay K 8644 binds to, and stabilizes, the open state of calcium channels and thus acts as a "calcium agonist" to mediate calcium-dependent cellular events such as catecholamine release from PC 12 cells.     

Effects of Angiotensin II on [3H]Noradrenaline Release and Phosphatidylinositol Hydrolysis in the Parietal Cortex and Locus Coeruleus of the Rat

Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.     

The metabolic pattern of spontaneously released [3H]norepinephrine in the presence of uptake inhibitors

The effect of uptake inhibitors on the spontaneous release of [3H]norepinephrine was studied using rat isolated vas deferens. Cocaine, desmethylimipramine, phenoxybenzamine, and guanethidine caused an increase in the spontaneous outflow of tritium, but did not alter the metabolic pattern of released [3H]norepinephrine. Bretylium caused an increase of both total tritium outflow and norepinephrine content. It is suggested that these drugs caused release of norepinephrine from storage sites and that norepinephrine was metabolized within the nerve terminals before reaching the biophase.     

3H]adrenaline release from hypothalamic synaptosomes and its modulation by clonidine: effects of chronic antidepressant drug regimens

[3H]Adrenaline ([3H]ADR, 40 nM) was accumulated by rat hypothalamic synaptosomes (P2) more rapidly and in significantly greater amounts than by similar preparations from cerebral cortex. There was no significant difference between these two tissues in the rate or amount of [3H]noradrenaline ([3H]NA, 40 nM) accumulation. Talusupram (10 microM), maximally inhibited the uptake of [3H]ADR into hypothalamic synaptosomes by 60%. Nomifensine further inhibited uptake by 14%. From these observations it was concluded that some [3H]ADR was accumulated into non adrenergic neuronal terminals. The effects of desipramine (DMI, 10 mg/kg/day and clorgyline (1 mg/kg/day) administration for 28 days on K+-evoked release of [3H]ADR was investigated using superfused hypothalamic synaptosomes. After both chronic antidepressant drug regimens, total [3H]ADR release (spontaneous + evoked) was significantly reduced. Evoked release of [3H]ADR (by KCl, 16 mM) was significantly reduced after the DMI but not the clorgyline regimens. Presynaptic alpha 2-adrenoceptor function in the hypothalamus was assessed during superfusion by measuring the reduction in K+-evoked release of [3H]ADR caused by clonidine (1 microM). The attenuating effects of clonidine on [3H]ADR release (42% in untreated controls and 36% after chronic clorgyline) was diminished (to 4%) after chronic DMI administration. Alpha 2 adrenoceptor numbers in the rat hypothalamus were not significantly changed after clorgyline or DMI administration, suggesting that the functional subsensitivity seen in synaptosomes after DMI, may not be related to alpha 2 adrenoceptor down regulation.     

Presynaptic muscarinic receptor activation enhances striatal dopamine release evoked by depolarization but not that induced by non-depolarizing stimuli

The release of [³H]dopamine stimulated by depolarization with 15 mM KCl of superfused rat striatal synaptosomes was potentiated by acetylcholine through the activation of presynaptic muscarinic receptors. In contrast, acetylcholine did not potentiate the release of [³H]dopamine elicited by d-amphetamine nor that caused by the calcium ionophore A23187. The dopamine carrier blocker nomifensine prevented the releasing action of amphetamine but not that of acetylcholine. The results suggest that the activation of muscarinic receptors on dopamine terminals in the rat corpus striatum selectively affects the calcium-dependent depolarization-induced release of the [³H]catecholamine. Moreover, the [³H]dopamine release caused by acetylcholine seems to occur independently of the membrane dopamine carrier.     

The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca²⁺]i, in bovine adrenal chromaffin cells was determined. At 10^–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca²⁺]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca²⁺]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca²⁺]i.     

Pharmacological effects of naltriben as a ligand for opioid mu and kappa receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.