Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

The origin of ovarian neuropeptide Y (NPY)-immunoreactive nerve fibres from the inferior mesenteric ganglion in the pig

Summary Applying a double-immunofluorescence technique, the porcine ovary is demonstrated to receive two populations of NPY-immunoreactive nerve fibres originating from the inferior mesenteric ganglion: one with colocalized tyrosine hydroxylase and supplying predominantly the ovarian vasculature, and a second, solely NPY-immunoreactive and almost exclusively associated with growing follicles. A third group of tyrosine hydroxylase-and dopamine--hydroxylase-positive, but NPY-negative nerve fibres is associated with ovarian blood vessels and, to a minor extent, with ovarian follicles. As revealed by retrograde tracing, the vast majority of postganglionic neurons projecting to the ovary is located in a discrete area of the ganglion, suggesting a somatotopic organization of the porcine inferior mesenteric ganglion. Moreover, the finding indicate that three subpopulations of postganglionic sympathetic neurons with different chemical codes supply different target components of the porcine ovary. The physiological relevance of the described neurons in the nervous control of ovarian functions remains to be elucidated.A portion of these results has been presented in abstract form (Majewski et al. 1991)     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

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.     

Ionic Requirements for Catecholamine Aggregating Actions on the Teleost Poecilia reticulata Melanophores

Norepinephrine (NE) and phenylephrine (Phe) were employed to study the ionic requirements for alpha adrenoceptor activation in the teleost Poecilia reticulata melanophores. As expected the beta adrenoceptor blocker, propranolol, increased the sensitivity of the preparation to NE (5.8 times), and was therefore employed in all the experimental procedures. Neither cocaine (a neuronal uptake blocker) nor dexamethasone (an extraneuronal uptake blocker) enhanced the sensitivity of the preparation to NE, suggesting that these inactivating mechanisms would not play a role in P. reticulata pigmentary system. However, in the absence of calcium, the dose-response curve (DRC) to NE was displaced to the left about 3.5 times, whereas the DRC to Phe was not affected. These results indicate that a neuronal uptake is active, but was not demonstrated by the classical pharmacological tools, probably due to an assymmetric display of the nervous endings. The DRC to NE was rightward displaced (14.1 times) in the presence of the calcium channel blocker Verapamil, whereas the DRC to Phe was not affected. These data suggest that P. reticulata melanophores possess a mixed population of alpha₁ and alpha₂ adrenoceptors, the activation of the latter eliciting an extracellular calcium influx. In sodium-free saline, the DRC to NE was rightward shifted (6.6 times) and the response to Phe was impaired in such a way that the maximal response was not achieved. The DRC to both NE and Phe were rightward displaced (7.9 and 2.7 times respectively) in the presence of the sodium channel blocker tetrodotoxin (TTX) 10^?7M. In potassium-free saline, the melanophore sensitivity to Phe was increased, whereas the responses to NE were not affected, suggesting a differential sensitivity of the two alpha adrenoceptor subtypes to the resulting membrane hyperpolarization. Based on the literature and on our data we propose that P. reticulata melanophores possess a mixed population of alpha₁ and alpha₂ receptors. The activation of both subtypes of alpha adrenoceptors elicits a Na⁺ ion influx through TTX-sensitive sodium channels. The stimulation of alpha₂ adrenoceptors also requires an extracellular calcium influx, through the opening of slow calcium channels.     

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.     

Insulin-Like Growth Factor-I-Enhanced Secretion Is Abolished in Protein Kinase C-Deficient Chromaffin Cells

Abstract: Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nM insulin-like growth factor-I (IGF-I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF-I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF-I on secretion from these cells. PKC was down-regulated in the cells by 16–18 h of treatment with β-phorbol didecanoate (β-PDD; 100 nM). Such treatment had no effect on high-K⁺-stimulated secretion from cells cultured without IGF-I; however, secretion from cells cultured with IGF-I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, α-PDD (100 nM), had no effect on secretion from untreated or IGF-I-treated chromaffin cells. The effect of β-PDD was time and concentration dependent, with 100 nM β-PDD producing a maximal effect in 8–10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by～40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both α-and ε-PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 μM) prevented the enhanced secretion normally seen in IGF-l-treated cells, whereas HA1004 had no effect. High-K⁺-stimulated ⁴⁵Ca²⁺ uptake in IGF-I-treated cells was attenuated by long-term treatment with β-PDD (200 nM) or H7 (100 μM). Together these observations suggest that PKC is required for IGF-I-enhanced secretion from chromaffin cells.