Héctor E. Chuluyan

Neuroendocrine integrative mechanisms in mammalian pineal gland: Effects of steroid and adenohypophysial hormones on melatonin synthesis in vitro

The time course for the decrease in norepinephrine concentration of rat pineal explants in culture indicated a significant fall starting at the 4th hour and completed after 16-24 h of incubation. Significant decreases of serotonin and 5-hydroxyindoleacetic acid (HIAA) levels in tissue, an increase of HIAA/serotonin ratio, and an increase of melatonin production rate in vitro were also observed as a function of the incubation time. Estradiol (10(-7)-10(-5) M) increased rat pineal melatonin content, testosterone (10(-5) M) decreased it and progesterone was devoid of activity when incubated with explants for up to 6 h. The in vitro stimulatory effect of estradiol on rat pineal methoxyindole synthesis was blocked by propranolol but not by phentolamine; propranolol also blocked the increase of nuclear estradiol-receptor complex produced by estrogen exposure of pineal explants. TSH (1-100 ng/ml), growth hormone (10-100 ng/ml) and LH (10 ng/ml) augmented rat pineal melatonin content while 100 ng/ml of FSH decreased it significantly. Prolactin exerted a biphasic effect on rat pineal explants, the lowest concentration augmenting melatonin content while the high concentration depressed it. Deep, intermediate and superficial segments of guinea-pig pineal glands showed an increase in melatonin concentration after a 6-h incubation in the presence of 10(-7)-10(-5) M estradiol.

Interactions of dendritic cells with fibronectin and endothelial cells.

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte‐derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte‐derived dendritic cells were obtained after culturing monocytes with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (800 U/ml) and interleukin‐4 (IL‐4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen‐DR (HLA‐DR) and CD86. On day 5 of incubation, 20–67% of moDC cultured in the presence of HS (HS‐moDC) expressed CD1a, b and c versus 94–97% when cultured in the presence of FBS (FBS‐moDC). DC showed a differential gradient of adhesion to FN: FBS‐moDC>HS‐moDC>sDC≈monocytes. Both FBS‐moDC and HS‐moDC were strongly positive for CD49e (α5‐integrin) and CD29 (β1‐integrin) but negative for CD49d (α4‐integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS‐moDC and HS‐moDC attached to endothelium (a 76% and 63% increase, respectively), only HS‐moDC were able to migrate through non‐activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS‐moDC are less adhesive to FN and endothelial cells but more motile than FBS‐moDC, and that α5β1‐integrin is the molecule involved in moDC adhesion to FN.

Release and effect ofγ-Aminobutyric acid (GABA) on rat pineal melatonin productionin vitro

Summary1.3H-γ-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pinealsin vitro.2.The release of3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+.3.In a Ca2+-free medium3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted.4.Norepinephrine (NE; 10−6–10−4M) stimulated3H-GABA release from rat pineal explants in a dose-dependent manner.5.The activity of 10−5M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (α1- andα1/α2-adrenoceptor blockers, respectively) and was unaffected by propranolol (β-adrenoceptor blocker).6.Theα1-adrenoceptor agonist phenylephrine (10−7-10−5M) and theβ-adrenoceptor agonist isoproterenol (10−5M) mimicked the GABA releasing activity of NE, while 10−7M isoproterenol failed to affect it; theα2-adrenoceptor agonist clonidine (10−7–10−5M) did not modify3H-GABA release.7.The addition of 10−4M GABA or of the GABA transminase inhibitorγ-acetylenic GABA or aminooxyacetic acid inhibited the melatonin content and/or release to the medium in rat pineal organotypic cultures.8.GABA at concentrations of 10−5M or greater partially inhibited the NE-induced increase in melatonin production by pineal explants.9.The depressant effect of GABA on melatonin production was inhibited by the GABA type A receptor antagonist bicuculline; bicuculline alone increased the pineal melatonin content. Baclofen, a GABA type B receptor agonist, did not affect the pineal melatonin content or release.10.The decrease in serotonin (5-HT) content of rat pineal explants brought about by NE was not modified by GABA; GABA by itself increased 5-HT levels.11.These results indicate that (a) GABA is released from rat pineals by a depolarizing stimulus of K+ through a mechanism which is partially Ca2+ dependent; (b) NE releases rat pineal GABA via interaction withα1-adrenoceptors; (c) GABA inhibits melatonin productionin vitro via interaction with GABA type A receptor sites; and (d) GABAs effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.

SLOWER GROWTH OF TUMOURS IN SYMPATHETICALLY DENERVATED MURINE SKIN

In order to examine tumour growth in sympathetically denervated murine skin, two breast cancer tumour lines were employed, i.e. M3 tumours, of a relatively high local growth and low metastatic capacity, and MM3-LN tumours, that grew locally at a slower rate but disseminated early to the lung. Mice subjected to unilateral superior cervical ganglionectomy or sham-operation 2 weeks earlier were used. M3 or MM3-LN tumours were implanted in the ipsilateral ear to the surgical procedure. Tumour size was assessed every 2-6 days, starting from the 7th day after tumour implantation. Growth of M3 and MM3-LN tumours was significantly slowed by a previous sympathetic denervation of the skin territory. There were no significant differences in the number or size of pulmonary metastases at autopsy between mice subjected to ganglionectomy or to sham-operation. Ganglionectomy increased significantly ipsilateral submaxillary lymph node ornithine decarboxylase activity by 62% and decreased noradrenaline content to 8% of the innervated contralateral lymph node. The present results indicate a local inhibitory modulation of tumour growth by the sympathetic nervous system.

Presynaptic effects of gamma-aminobutyric acid on norepinephrine release and uptake in rat pineal gland

The effect of τ-aminobutyric acid (GABA) on pineal norepinephrine (NE) release was examined in vitro in the rat pineal gland. Exposure of pineal expiants previously loaded with3H-NE to 1–100 μM GABA caused a dosedependent decrease of3H-NE release triggered by 60 mM K+, with a threshold GABA concentration of 1 μM and IC50 of about 10 μM. The inhibitory effect of GABA was mimicked by the type B GABA agonist baclofen, displaying a similar dose-response relationship as GABA. The type A GABA agonist muscimol increased depolarization-induced3H-NE release, while the co-incubation with GABA and the type A receptor antagonist bicuculline augmented significantly GABAs depressive effect on3H-NE release. Bicuculline alone brought about a significant decrease of3H-NE release. Neither GABA, nor baclofen, muscimol or bicuculline, modified the spontaneous pineal3H-NE efflux. Assessment of3H-NE uptake at a low NE concentration (0.5 μM) indicated that GABA decreased it in a dose-dependent manner (IC50=100 μM) through an effect blocked by bicuculline and mimicked by muscimol but not by baclofen; at a 5 μM-3H-NE concentration a bicuculline-sensitive GABA augmentation of uptake was found. A kinetic analysis study of the pineal NE uptake process indicated that GABA augmented both Vmax and Km of transmitter uptake. These results indicate that GABA may be a significant regulatory signal for rat pineal sympathetic synapses.

Serotonin release mechanisms in bovine pineal gland: stimulation by norepinephrine and dopamine.

An assessment of serotonin (5HT) release was made in bovine pineal gland. Bovine pineal fragments took up [3H]5HT by a Na+-dependent process exhibiting two apparent Km, i.e. a high affinity uptake system (Km = 220 nM) and a low affinity uptake system (Km = 197 microM). A significant release of [3H]5HT was elicited by increasing K+ concentrations in the medium (20-80 mM). Exposure of bovine pineal fragments to varying doses of catecholaminergic agonists indicated that a significant [3H]5HT release was elicited at the following threshold concentrations: 10(-6) M norepinephrine (NE), 10(-7) M dopamine (DA), 10(-6) phenylephrine and 10(-6) M isoproterenol. By employing specific receptor agonists and antagonists, the 5HT release activity of adrenergic agonists was found to be mediated by alpha 1-adrenoceptors, while that of DA by D2-dopaminergic receptors. 5HT release elicited by NE or DA, as well as that by 30 mM K+, was Ca2+-dependent. Both NE and DA increase 45Ca2+ uptake in a dispersed cell preparation of bovine pineal glands. As in the case of 5HT release, the effect of NE and DA on calcium uptake was mediated by alpha 1-adrenoceptors and D2-dopaminergic receptors, respectively. These results indicate that both NE and DA control 5HT release in bovine pineal gland.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti‐inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [3H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin‐2 (IL‐2) or OKT3 monoclonal antibodies in a dose‐dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI‐treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL‐4, IL‐6 and IL‐10 in the cell culture supernatants. SLPI‐treated monocyte culture supernatant inhibited the CD4+ lymphocyte proliferation but did not affect the proliferation of CD8+ cells. Moreover, IL‐2 increased T‐bet expression and the presence of SLPI significantly decreased it. Finally, SLPI‐treated monocyte culture supernatant dramatically decreased interferon‐γ but increased IL‐4, IL‐6 and IL‐10 in the presence of IL‐2‐treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

Presynaptic effects of melatonin on norepinephrine release and uptake in rat pineal gland

Abstract: The effect of melatonin injection on norepinephrine (NE) turnover rate in rat pineal gland was estimated from the decline of tissue NE levels after the injection of the tyrosine hydroxylase inhibitor α‐methyl‐p‐tyrosine. The administration of a single injection of 300 μg/Kg of melatonin at the beginning of the scotophase induced, 3 hr later, a significant decrease of pineal NE turnover. The possible direct effect of melatonin on pineal NE release was examined in vitro. Exposure of rat pineal expiants previously loaded with 3H‐NE to 10−8‐10−6 M melatonin decreased significantly 3H‐NE release triggered by 60 mM K+. This activity of melatonin was revealed only in pineals excised at night (0000 and 0400, i.e., at the fourth or eighth hours of darkness) and not in those excised in the middle (1400) or late light phase of the daily photoperiod (2000). Melatonin did not modify the spontaneous pineal 3H‐NE efflux. Melatonin decreased 3H‐NE uptake at a low NE concentration (0.5 μM) in a dose‐dependent manner (IC50≡ 10−10 M). A kinetic analysis of the pineal NE uptake process indicated that melatonin augmented both Vmax and Km of transmitter uptake. These results suggest that endogenously released melatonin may be a regultory signal for rat pineal sympathetic synapses.

Evidence suggesting that the sympathetic nervous system mediates thyroidal depression in turpentine-induced nonthyroidal illness syndrome.

Acute superior cervical ganglionectomy (SCGx) induces in the rat a supraliminal release of neurotransmitter in the innervated tissues (i.e., thyroid gland). This temporary adrenergic hyperactivity is correlated with a significant depression of the thyroid economy resembling the nonthyroidal illness (NTI) syndrome in the rat, and suggest that the sympathetic nervous system may mediate thyroidal changes in NTI. In order to gain further insight into the thyroidal depression in the NTI syndrome, we studied the thyroidal norepinephrine (NE) turnover in turpentine oil (TURP)-induced NTI syndrome and the role of the cervical ganglia (SCG) in the development of NTI in the rat. TURP administration to sham operated rats induced a rapid and significant fall in plasma T4 and TSH levels, in the thyroidal response to exogenous TSH (TIU) and in the thyroidal NE content compared to controls (sham + saline) (T4: 3.1 +/- 0.3 vs. 5.1 +/- 0.6 micrograms/dl, respectively, mean +/- SE, p less than 0.02; TSH: 1.4 +/- 0.4 vs. 4.7 +/- 1.4 ng/ml, respectively, p less than 0.05; TIU: 92 +/- 14 vs. 201 +/- 20 cpm.microliter thyroid/cpm.mg plasma (T/P ratio), respectively, p less than 0.01; thyroidal NE: 680 +/- 20 vs. 761 +/- 29 pg/mg thyroid, respectively, p less than 0.05). The thyroidal turnover rate of NE, however, was significantly increased in TURP-injected rats compared to controls (122 +/- 13 vs. 86 +/- 10 pg/mg/h, respectively, p less than 0.05). TURP injection to chronic SCGx rats induced a similar fall in plasma TSH compared to controls (SCGx + saline) (1.3 +/- 0.2 vs. 4.3 +/- 1.1 ng/ml, respectively, p less than 0.02); plasma T4 and TIU, however, did not change significantly (T4: 3.4 +/- 0.4 vs. 3.7 +/- 0.3 micrograms/dl, respectively, NS; TIU: 172 +/- 8 vs. 226 +/- 27 T/P ratio, respectively, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased pineal melatonin content coupled to restricted water availability in a Pavlovian conditioning paradigm in rats

The objective of this study was to assess whether rat pineal melatonin content could be modified in a classical conditioning paradigm. In rats kept under light (200 lux) from 06.00 to 18.00h daily, the time of lights off was selected as the unconditioned stimulus (US). Restricted water availability (from 10 min before to 10 min after light-dark, LD, transition) was the conditioned stimulus (CS). The conditioned and unconditioned responses were measured as the changes in pineal melatonin levels 4 h after LD transition. In animals under regular lighting conditions, lights out at 18.00 h (the US) caused a 4.4–7.8-fold increase of pineal melatonin concentration 4 h after later, when compared to animals maintained under light for the 4 h-period. After a training period of 7 days of restricted water availability (the CS), significantly augmented pineal melatonin levels were found in rats that were exposed to water but were maintained under light for the 4 h period after expected LD transition. The control animals for this experiment, i.e., rats which had undergone the training period, were kept for 4 h under light after expected LD transition, and did not receive water at LD transition, exhibited very low pineal melatonin levels. The conditioned increase of pineal melatonin content attained lower values than those in rats exposed to normal lighting conditions. It also fulfilled the contingency criterion, that is, it caused at trial a significant elevation of pineal melatonin content only when water availability was applied from 10 min previously to LD transition during training, and not 20 min after LD transition. After a training period of 7 days, restricted water availability applied 4 h before lights off (at 14.00 h), caused an enhanced production of melatonin 4 h later, regardless of the animals being exposed either to a dark or to a light environment. The results indicate that pineal melatonin production can be manipulated in a classical conditioning paradigm, when an appropriate CS stimulus is used.