The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro

The effect of various vertebrate somatostatins (SRIF) on basal growth hormone (GH) secretion from goldfish pituitary fragments was studied using an in vitro perifusion system. SRIF-14 caused a rapid and dose-dependent decrease in the rate of GH release from goldfish pituitary fragments. The half-maximal effective dose (ED50) of SRIF-14 was calculated as 1.3 nM following exposure to two minute pulses of increasing concentrations of SRIF-14, whereas the ED50 of SRIF-14 calculated after continuous exposure to sequentially increasing doses of SRIF-14 was 65 nM. This difference suggests that the pituitary fragments were less responsive to SRIF-14 in the latter experiment, possibly as a result of previous exposure to SRIF-14. SRIF-28 was found to be equipotent with SRIF-14 in decreasing basal GH secretion from the goldfish pituitary. In contrast, catfish SRIF-22, a uniquely teleost SRIF isolated from catfish pancreatic islets, did not alter GH secretion. These results provide further support for the hypothesis that SRIF-14 or a very similar molecule functions as a GH release-inhibiting factor in teleosts, indicating that this action of SRIF-14 has been fully conserved throughout vertebrate evolution.     

In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish

We studied the in vitro and in vivo effects of octanoylated goldfish ghrelin peptides (gGRL-19 and gGRL-12) on luteinizing hormone (LH) and growth hormone (GH) release in goldfish. gGRL-19 and gGRL-12 at picomolar doses stimulated LH and GH release from dispersed goldfish pituitary cells in perifusion and static incubation. Incubation of pituitary cells for 2 h with 10 nM gGRL-12 and 1 or 10 nM gGRL-19 increased LH-beta mRNA expression, whereas only 10 nM gGRL-19 increased GH mRNA expression. Somatostatin-14 abolished the stimulatory effects of ghrelin on GH release from dispersed pituitary cells in perifusion and static culture. The GH secretagogue receptor antagonist d-Lys(3)-GHRP-6 inhibited the ghrelin-induced LH release, whereas no effects were found on stimulation of GH release by ghrelin. Intracerebroventricular injection of 1 ng/g body wt of gGRL-19 or intraperitoneal injection of 100 ng/g body wt of gGRL-19 increased serum LH levels at 60 min after injection, whereas significant increases in GH levels were found at 15 and 30 min after these treatments. Our results indicate that, in addition to its potent stimulatory actions on GH release, goldfish ghrelin peptides have the novel function of stimulating LH release in goldfish.     

Studies on the prolactin-releasing mechanism of histones H2A and H2B

In previous studies we demonstrated that histone preparations possess multiple effects in vivo on pituitary hormone secretion. We have now studied the specificity and signal transduction pathways involved in the prolactin (PRL)-releasing activity of histones H2A and H2B on perifused and incubated rat pituitary cells. In the perifusion experiments, freshly dispersed pituitary cells were packed into short columns and were continuously perifused with serum-free medium. The substances to be tested (stimuli) were pumped through the perifusion circuit, at the end of which perifusate fractions were collected and PRL measured by specific RIA. In the incubation studies, freshly dispersed pituitary cells were incubated in a metabolic incubator with different stimuli at different doses and for varying times. Perifusion of cells with median eminence extract (1/30), histone H2A (30 microM) or histone H2B (30 microM), generated clear PRL release responses. Cells incubated with histone H2A and H2B showed a dose- and time-dependent stimulatory effect on PRL release which, for H2A, was blocked by peptide MB-35, an 86-120 amino acid synthetic fragment of histone H2A. The polycation, poly-lys was unable to mimic the action of histones. To detect the possible signal transduction pathways involved in the response of lactotrophs to histones, cells were incubated with the calcium ionophore A23187, the calcium chelator EGTA, the intracellular phosphoinositide enhancer LiCl, the intracellular cAMP enhancers caffeine, NaF and forskolin, and the protein kinase C inhibitor, trifluoperazine (TFP). Both EGTA (or EGTA plus A23187 ionophore) and TFP were able to reduce significantly the response of lactotrophs to histones. Our results confirm previous evidence that histones may act as hypophysotropic signals. The data also suggest that calcium- and diacylglycerol-associated pathways participate in these effects.     

The effect of gonadotropin-releasing hormone on growth hormone and gonadotropin subunit gene expression in the pituitary of goldfish, Carassius auratus

The goldfish brain contains at least two forms of gonadotropin-releasing hormone (GnRH): sGnRH and cGnRH-II. In goldfish sGnRH and cGnRH-II are present both in the brain and pituitary, and exert direct effects via specific GnRH receptors stimulating growth hormone (GH) and gonadotropin hormone (GtH) synthesis and secretion. In this study, we investigated the effects of sGnRH and cGnRH-II on GtH subunit (alpha, FSH-beta and LH-beta) and GH mRNA levels in the goldfish pituitary in vivo and in vitro. Injection of goldfish with sGnRH or cGnRH-II (4 microg/fish) stimulated GtH-alpha, FSH-beta and LH-beta mRNA levels after 24 h. For in vitro studies, goldfish pituitary fragments were treated continuously for 12 h with 10(-7) M sGnRH or cGnRH-II. Both sGnRH and cGnRH-II stimulated GtH-alpha, FSH-beta, LH-beta and GH mRNA levels, however, cGnRH-II appeared to have a more pronounced effect. Similar experiments were carried out using cultured dispersed goldfish pituitary cells. In this study, treatments for 12 h with 10(-7) M sGnRH or cGnRH-II also stimulated GtH and GH gene expression. The present results provide a basis for the investigation of the signal transduction pathways that mediate GnRH-induced changes in GtH subunit and GH mRNA levels in the goldfish pituitary.     

Age-related alterations in the stimulated release in vitro of catecholamines and luteinizing hormone-releasing hormone from the male rat hypothalamus

Using an in vitro perifusion system, the present study investigated the possibility that alterations in catecholamine and luteinizing hormone-releasing hormone (LHRH) secretion from the male rat mediobasal hypothalamus are present during the period of middle-age. The results indicate that, while tissue concentrations and baseline secretion of norepinephrine, dopamine and LHRH were similar between age groups, the patterns of dopamine and LHRH release in response to a series of depolarizing stimuli was different in the older animals. After all challenges, dopamine concentrations in the perifusate declined much more sharply for the middle-aged group, a finding that may be associated with a decrease with age in the pool of transmitter available for ready release. Also, tissue fragments from young adult rats were able to maintain the release of LHRH to a greater extent than tissue from the middle-aged animals, but only for the initial challenge period. The typical episodic pattern of LHRH release appeared to be disrupted in the older group following a second stimulus. It is possible that these age-related changes are early components of a dysruption in the hypothalamic mechanisms governing gonadotropin secretion.The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade name or commercial products constitute endorsement or recommendation for use.     

Comparison of hyposmolar and hyperosmolar effects on in vitro luteinizing hormone secretion by anterior pituitary cells

It has previously been described that perifusion of acutely dispersed adenohypophyseal cells with hypotonic medium causes an immediate high-amplitude "on" burst of luteinizing hormone (LH) secretion. In the present report the converse study with hyperosmolar solutions has been made. Perifusion with hypertonic medium depressed LH secretion; return to isotonicity caused an immediate high-amplitude "off" burst of LH secretion closely resembling that induced by hypotonic perifusion. The data give further support to the theory that exocytotic secretion may involve expansion of the outer cell membrane, thus drawing secretory granules to the cell surface where their contents are extruded.     

PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary

Gonadotropin-releasing hormone (GnRH) is produced by the hypothalamus and stimulates the synthesis and secretion of gonadotropin hormones. In addition, GnRH also stimulates the production and secretion of growth hormone (GH) in some fish species and in humans with certain clinical disorders. In the goldfish pituitary, GH secretion and gene expression are regulated by two endogenous forms of GnRH known as salmon GnRH and chicken GnRH-II. It is well established that PKC mediates GnRH-stimulated GH secretion in the goldfish pituitary. In contrast, the signal transduction of GnRH-induced GH gene expression has not been elucidated in any model system. In this study, we demonstrate, for the first time, the presence of novel and atypical PKC isoforms in the pituitary of a fish. Moreover, our results indicate that conventional PKC alpha is present selectively in GH-producing cells. Treatment of primary cultures of dispersed goldfish pituitary cells with PKC activators (phorbol ester or diacylglycerol analog) did not affect basal or GnRH-induced GH mRNA levels, and two different inhibitors of PKC (calphostin C and GF109203X) did not reduce the effects of GnRH on GH gene expression. Together, these results suggest that, in contrast to secretion, conventional and novel PKCs are not involved in GnRH-stimulated increases in GH mRNA levels in the goldfish pituitary. Instead, PD98059 inhibited GnRH-induced GH gene expression, suggesting that the ERK signaling pathway is involved. The results presented here provide novel insights into the functional specificity of GnRH-induced signaling and the regulation of GH gene expression.     

Stimulatory and inhibitory effects of progesterone on FSH secretion by the anterior pituitary.

The purpose of this study was to investigate whether progesterone exerted progesterone receptor mediated direct effects on the anterior pituitary in the secretion of FSH and whether such effects were mediated through the 5 alpha-reduction of progesterone. Treatment of anterior pituitary dispersed cells for 48 h with 0.5 nM estradiol reduced the ED50 for gonadotropin releasing hormone (GnRH)-stimulated FSH release from 0.58 to 0.36 ng/ml and the ED50 for GnRH-induced LH release from 0.54 to 0.19 ng/ml. When dispersed pituitary cells were treated with 0.5 nM estradiol and exposed to various doses of progesterone for 1 to 6 h, the most consistent rise in basal and GnRH-stimulated FSH release was observed with the 50 nM dose of progesterone with a 3-h exposure period. All three doses of progesterone elevated basal LH and GnRH-stimulated LH was increased by the 50 and 100 nM doses of progesterone during the 3-h period of treatment. Using the 50 nM dose of progesterone, basal and GnRH-stimulated LH was increased after 2, 3 and 6 h of progesterone treatment. When the period of exposure of progesterone was extended to 12, 36 or 48 h, there was a significant inhibition of GnRH-stimulated FSH release. GnRH-stimulated LH release was inhibited at 36 and 48 but not 12 h after progesterone treatment. These studies showed that the effect of progesterone administered for periods of 1 to 6 h enhanced the secretion of LH and FSH whereas progesterone administered for periods beyond 12 h inhibited FSH and LH release by dispersed pituitary cells in culture. These results are similar to those observed in vivo after progesterone treatment. Furthermore estrogen priming of the dispersed pituitary cells was necessary to observe the effects of progesterone. The progesterone antagonist RU486 prevented the progesterone-induced rise in GnRH-stimulated FSH release. Furthermore the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane- 17 beta-carboxamide also prevented the progesterone-induced rise in GnRH-stimulated FSH release in estrogen-treated dispersed pituitary cells. These results indicate that the anterior pituitary is a major site of action of progesterone in the release of FSH and that 5 alpha-reduction of progesterone plays an important role in FSH release.     

Role of PKC in the regulation of gonadotropin subunit mRNA levels: interaction with two native forms of gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproduction in all vertebrates through its actions on the production and secretion of pituitary gonadotropin hormones (GtHs). Most vertebrate species express at least two GnRHs, including one form, designated chicken (c)GnRH-II or type II GnRH, which has been well conserved throughout evolution. The goldfish brain and pituitary contain salmon GnRH and cGnRH-II. In goldfish, GnRH-induced luteinizing hormone (LH) secretion involves PKC; however, whether PKC mediates GnRH stimulation of GtH subunit mRNA levels is unknown. In this study, we used inhibitors and activators of PKC to examine its possible involvement in GnRH-induced increases in GtH-alpha, follicle-stimulating hormone (FSH)-beta and LH-beta mRNA levels in primary cultures of dispersed goldfish pituitary cells. Treatment with PKC inhibitors calphostin C and GF109203X unmasked a basal repression of GtH subunit mRNA levels by PKC; both inhibitors increased GtH subunit mRNA levels in a dose-dependent manner. PKC activators, 12-O-tetradecanoylphorbol 13-acetate (TPA), and 1,2-dioctanoyl-sn-glycerol, stimulated GtH subunit mRNA levels, whereas an inactive phorbol ester (4-alpha-TPA) was without effect. Thus, a dual, inhibitory and stimulatory, influence for PKC in the regulation of GtH subunit mRNA levels is suggested. In contrast, PKC inhibitor- and activator-induced effects were, for the most part, additive to those of GnRH, suggesting that conventional and novel PKCs are unlikely to be involved in GnRH-stimulated increases in GtH subunit mRNA levels. Our data illustrate major differences in the signal transduction of GnRH effects on GtH secretion and gene expression in the goldfish pituitary.     

Nicotine stimulates maturational gonadotropin (GtH2) release from carp (Cyprinus carpio L.) pituitary cells

1. Perifusion of dispersed pituitary cells and pituitary cell culture was used to investigate the effects of cholinergic drugs on the secretion of maturational gonadotropin (GtH₂) in carp.2. Nicotine strongly, and in a dose dependent manner, stimulated GtH₂ release in male and in female carp (from 10⁻⁸M in the Perifusion and 10⁻¹⁰M in the cells cultures).3. Nicotine is 10 times more active in females than in males.4. The results suggest that in carp, nicotine stimulates GtH₂ release directly from the pituitary cells, indicating a possible involvement of a cholinergic system in the regulation of GtH₂ secretion in teleost fish.     

Failure of 2 Hydroxyestradiol to interact with dopamine inhibition of human prolactin secretion in vitro and with dopamine receptors of prolactin-secreting adenomas

The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors.     

Selective depletion of dopamine in the goldfish pituitary caused by domperidone.

The effects of the dopamine type-2 receptor (D-2) antagonist domperidone on pituitary and brain amine concentrations and serum gonadotropin levels in the goldfish were investigated. Domperidone caused a long-lasting, dose-dependent depletion of dopamine in the goldfish pituitary. Pituitary concentrations of 5-hydroxytryptamine (5HT) were unaffected by domperidone treatment. Concentrations of noradenaline, dopamine, and 5HT in the hypothalamus and telencephalon were also unaffected by domperidone treatment. In contrast to the goldfish, dopamine levels in both mouse pituitary and hypothalamus were unaffected by domperidone treatment. The depletion of dopamine was observed in both sexually regressed and recrudescent, male and female fish, but elevation of serum gonadotropin levels in response to domperidone treatment occurred only in sexually recrudescent fish. Treatment of sexually recrudescent fish with the D-2 antagonists pimozide, (-)-sulpiride and eticlopride and the dopamine type-1 (D-1) antagonists SKF 83566 and SCH 23390 failed to elicit a depletion of pituitary dopamine or elevation of serum gonadotropin. Treatment of sexually recrudescent fish with domperidone, alpha-methyl-p-tyrosine or carbidopa elicited comparable depletions of pituitary dopamine and elevations of serum gonadotropin. The results suggest that in addition to D-2 receptor antagonist activity, domperidone has some other neuropharmacological action on dopaminergic neurones in the goldfish pituitary.     

The effects of pinealectomy on pituitary and plasma gonadotropin levels in Carassius auratus exposed to various photoperiod–temperature regimes

The effects of pinealectomy on pituitary and plasma gonadotropin levels and gonadal development in female goldfish exposed to various photoperiod-temperature regimes during different seasons were examined. Pinealectomy during autumn had no effect on either pituitary or plasma hormone levels or gonadal development. When goldfish are pinealectomized in spring and exposed to long photoperiod conditions, the ovaries regress and plasma gonadotropin levels are significantly depressed compared to sham operated animals. Sham operated goldfish exposed to short photoperiod conditions in spring had regressing ovaries whereas pinealectomized animals under this regime either spawned or had ovaries in the late vitellogenic phase. Plasma gonadotropin titres in the pinealectomized group were significantly lower than those of sham operated animals. The pineal can be either stimulatory or inhibitory to gonadal development depending on the photoperiod regime to which the animals are exposed. The pineal apparently influences gonadal activity by modulating gonadotropin secretion. A diurnal variation in plasma gonadotropin levels was also observed in both sham operated and pinealectomized goldfish exposed to a long photoperiod warm-temperature regime in spring.     

Estrogen and androgen elicit growth hormone release via dissimilar patterns of hypothalamic neuropeptide secretion

The dimorphic pattern of growth hormone (GH) secretion and somatic growth in male and female mammals is attributable to the gonadal steroids. Whether these hormones mediate their effects solely on hypothalamic neurons, on somatotropes or on both to evoke the gender-specific GH secretory patterns has not been fully elucidated. The purpose of this study was to determine the effects of 17beta-estradiol, testosterone and its metabolites on release of GH, GH-releasing hormone (GHRH) and somatostatin (SRIF) from bovine anterior pituitary cells and hypothalamic slices in an in vitro perifusion system. Physiological concentrations of testosterone and estradiol perifused directly to anterior pituitary cells did not affect GH releases; whereas, dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol increased GH. Perifusion of testosterone at a pulsatile rate, and its metabolites and estradiol at a constant rate to hypothalamic slices in series with anterior pituitary cells increased GH release. The androgenic hormones increased GHRH and SRIF release from hypothalamus; whereas, estradiol increased GHRH but decreased SRIF release. Our data show that estradiol and the androgens generated distinctly different patterns of GHRH and SRIF release, which in turn established gender-specific GH patterns.     

Development of an enzyme-linked immunosorbent assay for goldfish gonadotropin

An enzyme-linked immunosorbent assay (ELISA) for goldfish gonadotropin (GTH) was developed with the intent of devising a simple, reliable and nonradioisotopic assay for the measurement of GTH in goldfish biological samples. In this assay, soluble GTH of the standards or samples competes with carp GTH (cGTH) immobilized on a solid support (96-well microplate) for the fixation on antibodies to the beta-subunit of carp gonadotropin. The immobilized antigen-antibody complexes are then revealed by the peroxidase-antiperoxidase (PAP) technique. After revelation of the peroxidase activity, the absorbance value of each well is measured with a microplate reader. The cGTH concentration used for coating the wells is 2 ng/ml and the final dilution of the specific antibody is 1:80,000. The assay can be performed within 24 h and can be used over a range of 0.125-4 ng/ml. At about 50% binding, the intra- and interassay coefficients of variation are 5% and 9% respectively. The displacement curves generated by goldfish plasma or pituitary perifusion fractions were strictly parallel to the standard cGTH. In addition, the stimulation by salmon gonadotropin-releasing hormone of pituitary fractions perifused in vitro caused an immediate increase in the GTH measured in the collected fractions, strongly reinforcing the assumption that this assay indeed measures GTH.     

促性腺激素释放激素类似物对长臀鮠脑垂体促性腺激素分泌的影响

采用离体灌流孵育技术和促性腺激素的放射免疫测定方法,对长臀鮠(Cranoglanis bouderius)脑垂体碎片促性腺激素的分泌进行了研究。结果表明：持续的促性腺激素释放激素类似物(GnRH-A)能显著刺激退化期的长臀鮠离体脑垂体碎片促性腺激素(GTH)的分泌,并且长臀鮠脑垂体碎片对持续的GnRH-A刺激未表现出脱敏性,该结果与胡子鲇和鲇鱼相似,而与金鱼和鲤科鱼类不同;重复脉冲GnRH-A刺激对长臀鮠脑垂体碎片GTH分泌具有促进作用,而且存在剂量依存关系,与鲇鱼和鲤科鱼类相类似。上述结果表明在长臀鮠的人工繁殖中可以用持续高浓度GnRH-A刺激对长臀鮠进行催熟和催产。     

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin and somatolactin release from the goldfish pituitary in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in mediating growth hormone and gonadotropin release in the teleost pituitary. In the present study, we examined the immunohistochemical relationship between PACAP nerve fibers and prolactin (PRL)- and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibers with PACAP-like immunoreactivity (PACAP-LI) were identified in the neurohypophysis in close proximity to cells containing PRL-LI or SL-LI. Several cells with PRL-LI or SL-LI showed PACAP receptor (PAC(1)R)-LI. The cell immunoblot assay method was used to examine the effect of PACAP on PRL and SL release from dispersed goldfish pituitary cells. Treatment with PACAP increased the immunoblot area for PRL- and SL-LI from individual pituitary cells in a dose-dependent manner. The effect of PACAP on the expression of mRNAs for PRL and SL in cultured pituitary cells was also tested. Semiquantitative analysis revealed that the expression of SL mRNA, but not PRL mRNA, was increased significantly by the treatment with PACAP. The effect of PACAP on intracellular calcium mobilization in isolated pituitary cells was also investigated using confocal laser-scanning microscopy. The amplitude of Ca(2+) mobilization in individual cells showing PRL- or SL-LI was increased significantly following exposure of cells to PACAP. These results indicate that PACAP can potentially function as a hypophysiotropic factor mediating PRL and SL release in the goldfish pituitary.     

Long-term maintenance of growth hormone secretion in perifused rat anterior pituitary cells

We studied the effect of rat growth hormone-releasing factor-(1-43) acid, rGRF(1-43)OH, on the long-term secretion of rat growth hormone (rGH) in dispersed primary cultured cells of rat anterior pituitaries over a period of 7 days or longer. Results of the perifusion assay show that freshly dispersed cells secrete more rGH than 4-day-old redispersed cells (P less than 0.05), that a stabilization period ranging from 4 to 24 h allows a greater production of rGH per day than longer periods (P less than 0.05) and that the working concentrations of rGRF-(1-43)OH and prostaglandin E2 (PGE2) that insured the best responsiveness and longer viability are 50 pM and 10-1000 nM, respectively. Under these conditions, the cells continued secreting rGH after 42 days of perifusion, and 315 milligrams of rGH was produced over that period.     

Mechanisms for gonadotropin-releasing hormone potentiation of growth hormone rebound following norepinephrine inhibition in goldfish pituitary cells

In the goldfish, norepinephrine (NE) inhibits growth hormone (GH) secretion through activation of pituitary alpha(2)-adrenergic receptors. Interestingly, a GH rebound is observed after NE withdrawal, which can be markedly enhanced by prior exposure to gonadotropin-releasing hormone (GnRH). Here we examined the mechanisms responsible for GnRH potentiation of this "postinhibition" GH rebound. In goldfish pituitary cells, alpha(2)-adrenergic stimulation suppressed both basal and GnRH-induced GH mRNA expression, suggesting that a rise in GH synthesis induced by GnRH did not contribute to its potentiating effect. Using a column perifusion approach, GnRH given during NE treatment consistently enhanced the GH rebound following NE withdrawal. This potentiating effect was mimicked by activation of PKC and adenylate cyclase (AC) but not by induction of Ca(2+) entry through voltage-sensitive Ca(2+) channels (VSCC). Furthermore, GnRH-potentiated GH rebound could be alleviated by inactivation of PKC, removal of extracellular Ca(2+), blockade of VSCC, and inhibition of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII). Inactivation of AC and PKA, however, was not effective in this regard. These results, as a whole, suggest that GnRH potentiation of GH rebound following NE inhibition is mediated by PKC coupled to Ca(2+) entry through VSCC and subsequent activation of CaMKII. Apparently, the Ca(2+)-dependent cascades are involved in GH secretion during the rebound phase but are not essential for the initiation of GnRH potentiation. Since GnRH has been previously shown to have no effects on cAMP synthesis in goldfish pituitary cells, the involvement of cAMP-dependent mechanisms in GnRH potentiation is rather unlikely.     

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.