Effect of TNF-alpha on prolactin secretion from rat anterior pituitary and dopamine release from the hypothalamus: comparison with the effect of interleukin-1 beta.

The effects of human recombinant interleukin-1 beta and -6 and tumor necrosis factor-alpha (TNF-alpha) on the releases of PRL and dopamine were examined using monolayer cultures of rat pituitary cells and hypothalamic cells. The release of PRL from rat pituitary cells in 30 min was increased about 2-fold (p less than 0.05) by 10(5) U/l interleukin-1 beta, 10(5) U/l interleukin-6 or 100 micrograms/l TNF-alpha. TNF-alpha at 100 micrograms/l significantly increased PRL release within 5 min incubation and this effect continued throughout the next 30 min of incubation. Incubation for 5 min with TNF-alpha caused dose-dependent stimulation of PRL release. These cytokines did not modulate [3H]-dopamine release from primary cultures of hypothalamic cells. These results suggest that these cytokines stimulate PRL release directly at the pituitary gland, without modifying the release of dopamine from the hypothalamus.     

Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli.

The net percentage of release of arylsulfatase activity from purified rat mast cells induced by rabbit anti-rat F(ab')2 was consistently only about 1/3 that of histamine. Isoelectric focusing of the released and residual arylsulfatase activities demonstrated specific release of the A type without B and a net percentage of immunologic release of arylsulfatase A equivalent to that of histamine. When the net percentage of histamine and arylsulfatase A release were nearly maximal (88 and 76%) in response to the calcium ionophore A23187, specific release of arylsulfatase B did not occur. Thus, arylsulfatase A and not B was associated with the secretory granule released from the rat mast cell by reversed anaphylaxis or the calcium ionophore. In contrast, subcellular fractionation of water-lysed mast cells yielded arylsulfatase B with the heparin- and chymase-containing granule fraction and arylsulfatase A in the aqueous fraction comprised of cell sap and granule water eluate. It may be that arylsulfatase B resides in a minor second granule, whereas arylsulfatase A is loosely associated with the predominant secretory granule of the rat mast cell.     

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.     

Activin-A: a modulator of multiple types of anterior pituitary cells

When primary culture of rat pituitary cells were incubated with 1 nM activin-A for more than 24 hrs, activin-A significantly increased intracellular content of FSH without affecting the control of LH. Pretreatment of the cells with activin-A also enhanced LHRH-induced FSH release without affecting LH release. Furthermore, pretreatment of the cells with activin-A significantly reduced both GRF-mediated GH release and TRH-mediated PRL release. However, activin-A did not affect the response of ACTH and TSH to their releasing hormones. These results indicate that, in addition to the known action on gonadotrophs, activin-A also modifies the function of somatotrophs and lactotrophs.     

Studies in experimental animal models of the effect of ultraviolet radiation (UVC) on blood and isolated cell populations

The cellular and molecular basis of the therapeutically used effect of reinjected ultraviolet ( UVC ) irradiated blood is unknown. First approaches to that problem were made in this study by aid of model experiments. Neither the spontaneous degranulation nor the antigen-induced histamine release from rat connective tissue mast cells (in vivo) was influenced by the injection (i.v.) of ultraviolet irradiated blood or blood lymphocytes. By comparison of the effect of ultraviolet light on blood lymphocytes (number of dead cells, strength of chemoluminescence) after irradiation of the isolated cells and the unfractionated blood respectively, we could show that the strong light absorption within the blood sample prevents damage or functional alterations of the blood lymphocytes. The compound 48/80-induced histamine release from rat peritoneal mast cells can be completely inhibited by ultraviolet irradiation (0.6 mJ/cm2) without increasing the spontaneous histamine release.     

Cyclic adenosine 3',5'-monophosphate analogues modulate rat placental cell growth and differentiation

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-1-methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3',5'-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Differentiation-associated characteristics of rat placental cells were also affected by cAMP. cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.     

Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation

This study evaluated the effect of inhibitors of transmethylation on histamine release from rat mast cells and rat basophilic leukemia cells. IgE-mediated histamine release from rat basophilic leukemia cells (RBL-2H3 cells) was inhibited by 3-deazaadenosine (DZA) in the presence of L-homocysteine thiolactone (Hcy) or the combination of adenosine, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), and Hcy in a dose-dependent fashion. There were no significant changes in the cellular cAMP levels by these inhibitors. Histamine release induced by anti-IgE or dextran from normal rat mast cells was also blocked by DZA plus Hcy in a dose-dependent manner. DZA at 10(-3) M in the presence of 10(-4) M Hcy or the combination of 10(-3) M adenosine, 10(-4) M EHNA, and 10(-3) M Hcy inhibited lipid (perhaps phospholipid) methylation into RBL-2H3 cells without affecting choline incorporation. In the presence of 10(-3) M DZA plus 10(-4) M Hcy there was a 170-fold increase in [35S]AdoHcy with the concomitant appearance of 3-deaza-AdoHcy when the cells were incubated with [35S]methionine, thus indicating that these drugs inhibited methylation reaction(s) through the intracellular accumulation of AdoHcy and 3-deaza-AdoHcy. In contrast, histamine release from rat mast cells induced by the calcium ionophore A23187, compound 48/80, polymyxin B, or ATP was not inhibited by these compounds. These results suggest that IgE- or dextran-mediated histamine release involves methylation reactions(s), whereas the other secretagogues bypass this early step.     

Modulation of rat serosal mast cell biochemistry by in vivo dexamethasone administration

To examine steroid-induced biochemical alterations in the mast cell secretory process, rats were injected with intramuscular dexamethasone or saline for 4 days, and serosal mast cells and lung tissue were obtained from each group. Radioligand binding studies utilizing 1-[propyl-1,2-3H]dihydroalprenolol (3H-DHA) demonstrated a 23.1 +/- 0.8% increase in rat lung beta-adrenergic receptors in steroid-treated rats, but the mast cell beta-adrenergic receptors were unaffected. Neither resting mast cell cyclic adenosine 3':5'-monophosphate (cAMP) levels nor the degree of cAMP augmentation induced by isoproterenol were changed by steroid administration. Mast cells from rats treated with dexamethasone released only 48.6 +/- 8.9 and 58.8 +/- 6.0% of the beta-hexosaminidase released from saline-treated rat mast cells when sensitized with anti-dinitrophenyl (DNP) IgE and challenged with DNP-bovine serum albumin antigen or the calcium ionophore A23187, respectively. [3H]serotonin release in cells from steroid-treated rats was 41.8 +/- 7.9 and 87.6 +/- 2.6% of control release stimulated by antigen or A23187, respectively. [14C]arachidonic acid incorporation into mast cell phospholipids followed by antigen or A23187 challenge revealed that cells from dexamethasone-treated rats release 61.3 +/- 15.6% and 62.1 +/- 11.8% of labeled metabolites, respectively, compared to controls. The addition of exogenous arachidonic acid 5 min prior to antigen challenge caused a similar decrease in mediator release in cells from saline- and steroid-treated rats (36.7 +/- 6.1 and 38.4 +/- 0.9%, respectively). When arachidonic acid was added to sensitized cells after specific antigen, no significant changes in beta-hexosaminidase release were noted in either group. Chronic in vivo dexamethasone administration markedly decreases mast cell mediator release without changing resting cAMP levels. The release of arachidonic acid metabolites is reduced in steroid-treated cells, possibly through the inhibition of phospholipases. Exogenous arachidonic acid cannot overcome this inhibition, suggesting that an earlier step in phospholipid metabolism, perhaps involving phospholipase C, may be important.     

Biphasic effects of dexamethasone on growth hormone release in vitro

The effect of dexamethasone (Dex) on growth hormone (GH) release was examined in vitro in monolayer culture of normal rat pituitary cells and human somatotropinoma cells from patients with acromegaly. In either cell strain, Dex, at a concentration of 50 nM initially inhibited, but later (48 less than or equal to h) potentiated, the release of GH into the medium, with or without growth hormone releasing hormone (GHRH). The intracellular GH was significantly increased by 4-hour incubation with Dex in rat cell cultures. These results indicate a biphasic effect of glucocorticoids on GH release, irrespective of the origin of somatotrophs, and that the initial inhibitory effect is probably caused by inhibition of the release.     

The effect of clomiphene citrate on LH release by dispersed rat anterior pituitary cells

Preincubation of pituitary cells for 48 hours with estrogen or clomiphene citrate did not enhance LH release of dispersed pituitary cells after incubation for 4 hours without the secretagogues. In the presence of GnRH, estrogen increased release, but neither the racemic mixture of clomiphene nor its two trans and cis isomers had any appreciable effect on LH release. It is concluded that contrary to published reports, clomiphene in the rat pituitary may behave differently from estrogen in its effects on gonadotrphin release by dispersed cells.     

Magainin-2 releases histamine from rat mast cells.

The magainins are basic 23 amino acid peptides with a broad spectrum of antimicrobial activity. Their bactericidal effect has been attributed to their capacity to interact with lipid bilayer membranes. We observed histamine release by magainin-2 amide from rat peritoneal mast cells (ED50 = 13 micrograms/ml) but not from human basophils. This histamine-releasing reaction from peritoneal mast cells was due to a secretory rather than cytolytic effect, i.e., release occurred without concomitant liberation of lactic dehydrogenase. Furthermore, the pretreatment of mast cells with magainin-2 amide did not desensitize cells against subsequent challenge with other secretagogues. Maximum histamine release occurred in less than a minute at 25 and 37 degrees C. The addition of Ca2+ was not required for histamine release, although release was enhanced by the addition of 0.3-1 mM Ca2+. The addition of 3 mM Ca2+ or Mg2+ was markedly inhibitory. The presence of Na+ or Cl- ions in the medium was not required for release. Therefore, histamine release is not due to the formation of anion-selective channels in the membrane of mast cells. The results indicated that the characteristics of histamine secretion induced by magainin-2 amide were unlike IgE-mediated release but were similar to the mechanism of release attributed to some other basic peptides and to compound 48/80.     

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.     

Effect of dexamethasone on the release of pituitary hormones in monolayer cultures of rat pituitary cells

The effect of dexamethasone on the release of ACTH, GH, PRL, LH and TSH was studied in monolayer cultures of rat pituitary cells in 4-hour incubation. With or without the addition of rat hypothalamic extract, the release of GH was significantly inhibited by dexamethasone at concentrations higher than 10(-9) M, although less remarkably than that of ACTH. Intracellular ACTH and GH were unchanged. PRL, LH and TSH were not affected. These results indicate that dexamethasone, when exerted for 4 hours, suppressed the release of GH as well as ACTH, at least in part, at the pituitary level.     

Effects of rat serum on gonadotrophin release in vitro

Gonadotrophin release from a monolayer of cultured pituitary cells from the rat was increased by the addition of fresh rat serum or rat serum albumin to the culture medium. Similar effects were obtained when horse, chicken, lamb or porcine serum was used. Fetal bovine serum or bovine serum albumin showed a stimulatory effect on FSH and LH release but, unlike the case with rat serum, a dose-response release could not be demonstrated. In addition, fresh rat serum caused the accumulation of secretory granules in the gonadotrophs. The addition of LHRH to the culture medium also stimulated gonadotrophin release in a dose-response dependent manner. The dependence of the gonadotrophs on LHRH was markedly increased by the addition of 50% fresh rat serum to the culture medium. These results indicate that fresh rat serum contains some unknown factor(s) which 1) stimulates gonadotrophin release, 2) causes an accumulation of secretory granules, and 3) is important for maintaining secretory capacity of gonadotrophs.     

Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture.     

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1 min, and decayed to basal values in 10 min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca²⁺ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1^?/? rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.     

Stimulation of insulin release by L-glutamine

Summary Under normal environmental conditions, L-glutamine is well oxidized but fails to stimulate insulin release in rat pancreatic islets. However, a marked stimulation of insulin release by L-glutamine, without alteration in its oxidation rate, occurs when the intracellular pH of the islet cells is decreased and/or when theophylline is added to the incubation medium.     

Dissimilar responsiveness of cultured corticotrophs and melanotrophs to tripeptide aldehydes

Cultured cells from adult rat anterior pituitaries or intermediate lobes were treated with the proteinase inhibitor tripeptide aldehydes BOC-DPhe-Pro-Arg-H (Boc-fPRH) and DPhe-Pro-Arg-H (fPRH), ovine corticotropin-releasing factor (oCRF), and bromocriptine. One millimolar fPRH stimulated basal, and slightly enhanced oCRF-induced ACTH release by melanotrophs in short-term experiments. The basal release of alpha-MSH was also stimulated by the drug. In long-term experiments, fPRH elevated markedly both the release and the intracellular level of ACTH; BOC-fPRH caused an increased alpha-MSH release. Tritiated fPRH had no preference for POMC-producing cells and BOC-fPRH or fPRH were harmless to the cell morphology. In anterior pituitary cell cultures, fPRH diminished slightly basal and oCRF-induced ACTH release. Bromocriptine was ineffective on corticotrophs, however, in melanotrophs it inhibited ACTH release markedly with or without fPRH in the medium. The dissimilar responsiveness of the corticotrophs and melanotrophs to the peptide aldehydes may be interpreted in terms of their differing membrane receptors or intracellular mechanism of stimulus-secretion coupling.     

Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone

Osteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate-resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 micrograms/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.