In vitro study of immunoreactive corticotropin-releasing factor release from the rat hypothalamus

Immunoreactive corticotropin-releasing factor (I-CRF) release from rat hypothalami was studied in vitro utilizing a perifusion of rat hypothalami and a rat CRF RIA. Basal release of I-CRF from the hypothalamus of adrenalectomized or hypophysectomized rats was higher than in that of normal rats. K+-induced I-CRF release was completely suppressed by omission of Ca++ from the medium. Dexamethasone suppressed I-CRF release from hypothalami, but not from median eminence (ME). C-AMP and angiotensin II had mild stimulatory effects on I-CRF release. These results suggest that 1) the feedback mechanism acts mainly on a higher level than ME, and 2) c-AMP and angiotensin II may be involved in CRF-releasing mechanism(s).     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

Inhibitory effect of norepinephrine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of catecholamines on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Norepinephrine had a potent inhibitory effect on I-CRF release in a dose-dependent manner at 0.1 nM-1 microM concentrations, but dopamine did not. This inhibitory effect of norepinephrine was completely blocked by propranolol, but only partially blocked by phentolamine. Isoproterenol also had a potent inhibitory effect at 0.01-100 nM concentrations, and a high dose of phenylephrine (10 nM) inhibited I-CRF release. Clonidine did not influence I-CRF release. These results suggest that norepinephrine inhibits I-CRF release mainly through the beta-adrenergic receptor and partially through the alpha 1-receptor.     

Regulation of Bcl-2 Protein Expression in Human Neuroblastoma SH-SY5Y Cells: Positive and Negative Effects of Protein Kinases C and A, Respectively

Abstract: Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [³H]dopamine-loaded rat striatal slices. Our results show that illumination of RBS-pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µ M ), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2-bis( o -aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 10 µ M ) had no effect on the dopamine release stimulated by illumination of RBS-pretreated slices. The concentration of BAPTA-AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µ M ). Superfusion with zaprinast (10 µ M ) had no effect on RBS-induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.     

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.     

Ethanol-Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates

The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the delta-opiate receptor agonists Met5-enkephalin, [D-Ala2,D-Leu5]enkephalin (DADLE), and [D-Ser2]Leu-enkephalin-Thr (DSLET), increased the basal DA release without affecting potassium-stimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) and the epsilon-opioid agonist beta-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective delta-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous delta-opiate agonist.     

Potassium-Induced Release of Endogenous Morphine from Rat Brain Slices

Abstract: Endogenous morphine has been clearly demonstrated by gas chromatography/mass spectrometry in the brain, spinal fluid, adrenal glands, and liver of mammals. To clarify the role of endogenous morphine, its release from rat brain slices was studied in vitro in the presence of high potassium concentrations, with and without calcium in the medium. The perfusate was hydrolyzed, solid phase-extracted, and then analyzed by gas chromatography/mass spectrometry. Depolarization due to high potassium concentrations increased the release of the alkaloid manyfold with respect to the basal value, and the release was dependent on the presence of calcium in the medium. These results suggest that endogenous morphine might act as a neurotransmitter or neuromodulator in the rat CNS.     

Inhibition of prolactin and growth hormone secretion by nickel

Nickel (Ni++) is a potent inhibitor of prolactin (PRL) secretion from isolated rat pituitary quarters in vitro, suppressing both basal PRL release and the stimulation of PRL secretion due to theophylline and dibutyryl cyclic AMP. Stimulation of growth hormone (GH) secretion by synthetic GHRH is also blunted by Ni++, although basal GH release and stimulated GH release due to theophylline or dibutyryl cyclic AMP are not suppressed. Ni++ antagonizes the stimulation of both PRL and GH secretion by barium (Ba++) ion, suggesting that the inhibitory effects of Ni++ on hormone release are due to an antagonism of calcium uptake or redistribution.     

Store-operated calcium channels in HL-60 cells effects of temperature,differentiation and loperamide

The effect of temperature on calcium release and influx has been compared in differentiated and undifferentiated HL-60 cells. Receptor-mediated release of intracellular calcium by ATP was little affected by temperature in HL-60 cells. In differentiated HL-60 cells the store-operated calcium (SOC) channel-dependent sustained elevation of calcium levels after ATP was maximal at 25-29 degrees C; at higher temperatures calcium levels returned relatively rapidly towards basal levels. In undifferentiated cells, a SOC channel-dependent sustained elevation of calcium levels was not observed with levels returning to basal levels much more rapidly than in differentiated cells. The initial thapsigargin-initiated elevation of calcium did not become maximal until about 25 degrees C in both differentiated and undifferentiated HL-60 cells. In differentiated cells, the SOC channel-dependent sustained elevation of calcium after thapsigargin was maximal at 30-37 degrees C, while in undifferentiated cells, the sustained elevation was maximal at 25-30 degrees C. Loperamide, which augments the SOC channel-dependent sustained elevation of calcium, showed a temperature-dependent response that was maximal at about 22 degrees C after either ATP or thapsigargin and was minimal at 37 degrees C. In contrast, inhibition of SOC channel-dependent elevation of calcium by miconazole or trifluoperazine was not greatly affected by temperature.     

Hormonal stimulation of proline synthesis in the fat body of the fruit beetle, Pachnoda sinuata, is calcium dependent.

The role of calcium in the transduction of the hyperprolinaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata using in vivo and in vitro methods to measure changes in the concentration of proline and its precursor alanine. Extracellular calcium is necessary for maximal stimulation of proline synthesis at saturating doses of Mem-CC (0.3 nM) in vitro. This effect depends on the dose of Ca(2+): maximal proline synthesis of 2.1 micromol mg(-1) protein h(-1) was stimulated by Mem-CC at calcium levels of 0.5 mM, and the EC(50) was 0.16 mM. Using the ionophore A 23187 in vivo and in vitro, we demonstrated that the extracellular calcium acts, via an influx into the cell, on the stimulation of proline production and alanine consumption. The release of calcium from intracellular sources is part of the signalling process: the agent thapsigargin, which inhibits the Ca(2+)-ATPase, is able to stimulate proline synthesis in vivo and in vitro. Thimerosal, however, which triggers the release of calcium from IP3-sensitive stores in the endoplasmic reticulum, had no influence on proline production nor alanine consumption, indicating that inositolphosphates are not part of the transduction of the hyperprolinaemic signal of Mem-CC. Both substances, thapsigargin and thimerosal, stimulate calcium entry in vitro from the medium (similar to Mem-CC), which indicates that a capacitative calcium entry takes place. Neither the entry of extracellular calcium nor the release from the endoplasmic reticulum, however, are alone sufficient for a full stimulation of proline synthesis in vitro. The results of the present study suggest that calcium from extra- as well as from intracellular sources is part of the second messenger system for the transduction of the hyperprolinaemic signal of Mem-CC in the fat body of P. sinuata. Calcium acts most likely via the elevation of cAMP levels: the concentration of this cyclic nucleotide in the fat body during in vitro incubation was elevated by 487% by Mem-CC in the presence of calcium, while the increase was only 122% when calcium was absent.     

Synthesis and biological activity of oxo-7H-benzo[e]perimidine-4-carboxylic acid derivatives as potent, nonpeptide corticotropin releasing factor (CRF) receptor antagonists

A novel series of derivatives of oxo-7H-benzo[e]perimidine-4-carboxylic acid (I) potently displaced radioligand binding of 125I-CRF to both CRF1 and CRF2 receptors. The members of this series antagonized CRF-stimulated cAMP formation and CRF-stimulated corticotropin release from rat pituitary in vivo. These are the first nonpeptide antagonists to show activity at both CRF1 and CRF2 receptors.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.     

Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats.

Using a microdialysis method, we have investigated effects of the voltage-dependent calcium channel blockers, verapamil, nicardipine, omega-conotoxin and flunarizine on the dopamine release and metabolism in the striatum of freely moving rat. Perfusion of verapamil (1-300 microM) and nicardipine (1-100 microM), an L-type calcium channel blocker, into the striatum through the dialysis membrane showed a dose-dependent decrease of dopamine release in the dialysate and slight increase of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Treatment of omega-conotoxin (0.1, 1 microM), an N-type channel blocker, decreased about 50% basal dopamine release and slightly decreased DOPAC and HVA levels. Treatment with flunarizine (10 microM), an T-type channel blocker, did not affect the dopamine release and metabolism. From these data, it appears that treatments of the L- and N-type voltage-dependent calcium channel blockers in rat striatum suppress basal dopamine release, but T-type blocker does not suppress it, suggesting that L-, N- and T-type calcium channels regulate in vivo dopamine release in a different mechanism.     

Localization of calcium in the cyanobiont and gonidial zone ofCycas revoluta Thunb. by microelectrodes,chlorotetracycline, electron spectroscopic imaging and electron energy loss spectroscopy

Summary Ionic calcium concentration was measured in the gonidial zone of fresh coralloid roots by means of calcium microelectrodes. It was 10⁻⁶ M in the apical segments of coralloid roots and increased to 10⁻⁵ M in the gonidial zones of median and basal segments. Loosely membrane-bound calcium was evidenced by using chlorotetracycline (CTC) or ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and CTC, in cell walls of columnar cells ofCycas and in the cytoplasm of cyanobiont. Sub-cellular localization of calcium was obtained by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) analyses applied at transmission electron microscopy on thin, unstained sections of gonidial zone of coralloid roots. By means of these techniques, bound-calcium was detected inside the mucilage of apical and median segments whereas, in the basal segments, it was completely absent. In the heterocysts of apical segments of coralloid, calcium was localized on the envelope, cell walls, thylakoids and cyanophycin granules. In the gonidial zone of the basal segments, dead or degenerating heterocysts completely lacked calcium. Therefore, the high ionic calcium amounts detected in the gonidial zone of median and basal segments could represent a minor calcium uptake by the cells or release by lysed ones. The decreases in nitrogenase activity recorded in the median and basal segments of the coralloid roots paralleled the decrease in calcium amount in heterocyst envelope.     

The effects of pentachlorophenol, phenol and other pollutants on the liver of carp (Cyprinus carpio L.)

Liver tissue of carp was kept in roller tubes and the basal and epinephrine-induced release of glucose after 6 or 24 hr incubation were measured. The amount of liver glycogen after incubation was also determined. The liver was taken from carp treated in vivo with pollutants, mainly PCP or phenol, or was exposed to these pollutants in vitro. Treatment of carp in vivo with 10-10(4) micrograms/l phenol reduced the basal and the epinephrine-induced release of glucose from the liver. Treatment with low doses increased the glycogen content of the liver slightly, treatment with higher doses reduced the amount. Treatment of carp with low doses of several pollutants decreased mainly the basal glucose release from the liver and reduced the glycogen content. In vitro incubation of the liver with PCP or phenol for 3 days reduced at first the basal release and later the epinephrine-stimulated release of glucose from the liver. After a few days the glycogen content of liver exposed to pollutants was more strongly reduced than that of controls. The phosphorylase activity was slightly increased in liver tissue by the pollutants.     

Restoration of responsiveness to gonadotropin releasing hormone (GnRH) in calcium-depleted rat pituitary cells.

GnRH-stimulated, but not basal, luteinizing hormone (LH) release from cultured pituitary cells requires extra-cellular calcium. The present studies were designed to show whether cells which had lost responsiveness to GnRH in the absence of extracellular calcium (“Ca²⁺-depleted cells”) could regain responsiveness by readdition of calcium to the media. The addition of calcium-containing medium to cells which were preincubated (75 min) in calcium-free medium resulted in elevated basal LH release. Addition of GnRH to the media in the presence of calcium did not cause additional stimulation of LH release above the elevated basal level. Incubation of Ca²⁺-depleted cells in calcium-containing media for 2 h before measuring responsiveness depressed the basal level to near that seen in control cells and GnRH was able to stimulate LH release, but not to as high a level as in control cells (which were preincubated in 1 mM Ca²⁺-containing media). After incubation of calcium depleted cells in calcium-containing media for 3 h or 5 h, the basal and stimulated levels of LH response were statistically indistinguishable from those seen in control cells.     

Calcium ions as a mediator in GnRH action on gonadotropin release in the common carp (Cyprinus carpio L)

Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.     

ACTH inhibits the capsaicin-evoked release of CGRP from rat adrenal afferent nerves

The adrenal cortex is innervated by afferent fibers that have been implicated in affecting cortical steroidogenesis. Modulation of neurotransmitter release from afferents may represent a regulatory system for the control of adrenal cortical function. The present studies validate an in vitro superfusion technique for adrenal capsules employing the drug capsaicin, which activates a subset of afferent fibers and induces the release of calcitonin gene-related peptide (CGRP). Capsaicin-evoked CGRP release from adrenal afferents was blocked by capsazepine, a competitive antagonist for the capsaicin receptor, or by removal of extracellular calcium. Exogenous ACTH prevented capsaicin-evoked CGRP release, elevated basal aldosterone release, and prevented capsaicin-induced reduction in aldosterone release. Immunolabeling for the recently cloned capsaicin vanilloid receptor 1 demonstrated its presence in adrenal nerves. These results show that in vitro superfusion of adrenal capsules can be used to characterize factors that modulate neurotransmitter release from adrenal afferents. Furthermore, the results suggest that activation of adrenal afferents in vivo may attenuate aldosterone steroidogenesis and that high levels of ACTH may prevent this phenomenon.     

Development of Transmitter-Releasing Capacity in Neuron-Enriched Tissue Cultures

Dissociated cell cultures derived from whole brains of foetal rats (17 days of gestation) were maintained for periods of up to 21 days in vitro for the purpose of studying the transmitter-releasing properties of the dopaminergic neuronal cells and glial cells. In the neuron-enriched cultures, after 3 days in vitro, [3H]dopamine was released in response to depolarizing stimuli. Both the potassium and veratrine-evoked release of dopamine was Ca2+ dependent. Veratrine-evoked release was reduced in the presence of the calcium channel blocker verapamil and was tetrodotoxin sensitive. Glial cultures, after 7 days in vitro, did not respond to any depolarizing stimuli, although they displayed a significant ability to take up [3H]dopamine. Comparison between static incubations and perfused cultures showed no difference in the patterns of release resulting from veratrine stimulation. Tyrosine hydroxylase activity increased progressively in neuron-enriched cultures but was not detectable in glial cultures. These results show that neuron-enriched cultures respond to depolarizing stimuli in a manner similar to excised adult basal ganglia tissue, with the appearance of functional ionic channels after 3 days in vitro.     

Stage-specific production and release of juvenile hormone esterase from the ovary of Diploptera punctata

Juvenile hormone esterase (JHE) is a catabolic enzyme that specifically degrades juvenile hormone (JH) and has been identified in hemolymph and tissues in both larvae and adults of numerous insect species. This study investigates the presence of JHE in ovaries of the viviparous cockroach, Diploptera punctata, and the in vitro release of JHE from these ovaries during the first gonadotrophic cycle. JHE is released in vitro from maturing basal (most posterior) follicles and from follicle cells isolated from oocytes during the short period of time between spermatophore release and chorion formation. Enzyme release is dependent upon the presence of calcium in the medium. This released ovarian JHE appears to be larger than and to display ionic characteristics that are different from the isolated hemolymph and fat body JHEs. In addition, JHE activity measured in homogenates of whole ovaries and subsequently oviposited basal oocytes increases dramatically following spermatophore release, coincident with a previously described decline in JH titer in the ovary. A likely role for ovarian JHE is the site-specific degradation of JH in and around the oocyte prior to fertilization and embryonic development.