RESUMO

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 approximately monocytes. Both FBS-moDC and HS-moDC were strongly positive for CD49e (alpha5-integrin) and CD29 (beta1-integrin) but negative for CD49d (alpha4-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 alpha5beta1-integrin is the molecule involved in moDC adhesion to FN.

Assuntos

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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 18.00 h 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.

Assuntos

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The presence of gamma-aminobutyric acid (GABA) and peripheral and central benzodiazepine receptors in the mammalian pineal gland prompted the examination of GABAergic transmission in dispersed bovine pineal cells. The effect of GABA on 3H-serotonin (5HT) release was examined in bovine pineal cells. GABA, by acting through GABA B receptor subtype, decreased 5HT release and by acting through GABA B, and presumably through GABA A receptor subtypes, inhibited depolarization-induced 45Ca2+ uptake in bovine pinealocytes. GABA, by acting on GABA B as well as on GABA A receptors, prevented the 5HT2- or 5HT1C-mediated stimulatory effect of serotonergic agonists on calcium uptake in pineal cells. GABA augmented 36Cl- uptake by bovine pineal cells. These results are interpreted to indicate that, by regulating the release of 5HT, GABA may modulate the synthesis and action of melatonin.

Assuntos

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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)

Assuntos

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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.

Assuntos

RESUMO

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 alpha-methyl-p-tyrosine. The administration of a single injection of 300 micrograms/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 explants 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 microM) in a dose-dependent manner (IC50 identical to 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 regulatory signal for rat pineal sympathetic synapses.

Assuntos

RESUMO

GABA is present in the pineal gland of several mammals, where it is synthesized in situ as well as taken up from the circulation. This article reviews available information suggesting a local, physiological role of pineal GABA. Both the pinealocytes and the glial pineal cells have the capacity to take up GABA from the extracellular space. The GABA synthesizing enzyme glutamic decarboxylase (GAD) is detectable in the pineal gland; in the bovine pineal GAD exhibits "neuronal-like" properties. By employing a specific antibody against GABA, about 15% of pinealocytes gave a positive reaction in bovine pineal glands. After a depolarizing stimulus, GABA was released from bovine and rat pineal glands by both Ca2(+)-dependent and Ca2(+)-independent processes. By employing neuronal and glial GABA uptake inhibitors, most 3H-GABA release in bovine pineal gland could be attributed to a "neuronal" (presumably pinealocyte) compartment. Several components of the GABA type A receptor supramolecular complex (i.e., GABA binding sites, central-type benzodiazepine binding sites, Cl- ionophore), as well as a minor population of GABA type B receptor sites, were detected in bovine and human pineal glands. In the rat pineals, GABA is released by norepinephrine (NE) acting through alpha 1-adrenoceptors. Physiological concentrations of GABA, by its effect on type A receptor sites, impaired NE-induced melatonin release; by acting on GABA type B receptors, it decreased NE release. Another presumable presynaptic effect of GABA (i.e., to augment maximal velocity and to decrease affinity of NE uptake) was mediated by type A receptor sites. It is proposed that pre- and postsynaptic activity of GABA in the pineal does not differ from that found for GABA interneurons in local circuits of the brain.

Assuntos

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In order to analyze whether the bovine pineal gland is a homogeneous or a heterogeneous structure as far as monoamine content, the regional differences in norepinephrine (NE), dopamine (DA), serotonin (5HT), and 5-hydroxyindoleacetic acid (5HIAA) contents were assessed by high-pressure liquid chromatography. NE content was maximal in the proximal (close to the recessus pinealis) region and decreased in a rostral-caudal direction to achieve minimal values at the distal region. DA exhibited an opposite trend to NE, NE/DA ratios varying from 3.2 (proximal region) to 1.4 (distal region). Significantly lower NE content was found at the inferior as compared to the superior pineal region, and at the cortex as compared to the medulla. No significant differences were detected in DA concentration of these latter pineal regions, or in 5HT or 5HIAA concentration as a function of the region examined. 3H-5HT and 3H-NE uptake were maximal at the proximal zone in a rostral-caudal direction, at the superior as compared to the inferior region, and at the medulla as compared to the cortex. Unlabeled NE was equally effective to compete with 3H-5HT uptake in the several pineal regions studied. While NE increased maximally 3H-5HT release in a rostral-caudal direction, DA exhibited an opposite trend, displaying maximal 5HT release activity at the distal pineal region. DA and NE 5HT-releasing activity were greater in the pineal medulla than in the cortex, and did not exhibit differences in the superior as compared to the inferior pineal aspects. Excess (55 mM) K+ released 3H-5HT to a similar extent regardless of the pineal region examined.

Assuntos

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The effect of tau-aminobutyric acid (GABA) on pineal norepinephrine (NE) release was examined in vitro in the rat pineal gland. Exposure of pineal explants previously loaded with 3H-NE to 1-100 microM GABA caused a dose-dependent decrease of 3H-NE release triggered by 60 mM K+, with a threshold GABA concentration of 1 microM and IC50 of about 10 microM. 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-induced 3H-NE release, while the co-incubation with GABA and the type A receptor antagonist bicuculline augmented significantly GABA's depressive effect on 3H-NE release. Bicuculline alone brought about a significant decrease of 3H-NE release. Neither GABA, nor baclofen, muscimol or bicuculline, modified the spontaneous pineal 3H-NE efflux. Assessment of 3H-NE uptake at a low NE concentration (0.5 microM) indicated that GABA decreased it in a dose-dependent manner (IC50 = 100 microM) through an effect blocked by bicuculline and mimicked by muscimol but not by baclofen; at a 5 microM-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.

Assuntos

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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.

Assuntos

RESUMO

1. 3H-gamma-Aminobutyric acid (GABA) release elicited by a depolarizing K+ stimulus or by noradrenergic transmitter was examined in rat pineals in vitro. 2. The release of 3H-GABA was detectable at a 20 mM K+ concentration in medium and increased steadily up to 80 mM K+. 3. In a Ca2+-free medium 3H-GABA release elicited by 30 mM K+, but not that elicited by 50 mM K+, became blunted. 4. Norepinephrine (NE; 10(-6)-10(-4) M) stimulated 3H-GABA release from rat pineal explants in a dose-dependent manner. 5. The activity of 10(-5) M NE on pineal GABA release was suppressed by equimolecular amounts of prazosin or phentolamine (alpha 1- and alpha 1/alpha 2-adrenoceptor blockers, respectively) and was unaffected by propranolol (beta-adrenoceptor blocker). 6. The alpha 1-adrenoceptor agonist phenylephrine (10(-7)-10(-5) M) and the beta-adrenoceptor agonist isoproterenol (10(-5) M) mimicked the GABA releasing activity of NE, while 10(-7) M isoproterenol failed to affect it; the alpha 2-adrenoceptor agonist clonidine (10(-7)-10(-5) M) did not modify 3H-GABA release. 7. The addition of 10(-4) M GABA or of the GABA transaminase inhibitor gamma-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(-5) M 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 with alpha 1-adrenoceptors; (c) GABA inhibits melatonin production in vitro via interaction with GABA type A receptor sites; and (d) GABA's effect on NE-induced melatonin release does not correlate with the lack of effect on the NE-induced decrease in pineal 5-HT content.

Assuntos

RESUMO

The effect of local sympathetic denervation on immune responses of submaxillary lymph nodes (SmLN) of mice was examined in animals subjected to unilateral superior cervical ganglionectomy (SCGx). Norepinephrine (NE) content in ipsilateral SmLN decreased by 90% 7-20 days after SCGx, while bilateral SCGx resulted in a 91% decrease of SmLN NE content. SmLN of mice subjected to unilateral SCGx exhibited greater plaque-forming cell (PFC) response than the innervated contralateral SmLN, when challenged i.d. or i.p. with sheep red blood cells (SRBC) 10-20 days after surgery. In mice challenged with SRBC at an early phase of sympathetic nerve paralysis (i.e., 2 h after SCGx), PFC response was already increased in the ipsilateral SmLN whereas during the anterograde degeneration of nerve endings (6-24 h after SCGx) an impending depression of PFC number was observed. Contact hypersensitivity and allogeneic delayed-type reactions were also enhanced in the ear ipsilateral to SCGx. Primed SmLN cells of nodes located ipsilaterally to SCGx, when injected to (BALB/c x C57BL/6) F1, resulted in significantly higher spleen index than similarly treated, contralateral SmLN. In contrast, mitogenic stimulation under various experimental protocols failed to reveal significant differences between proliferation of cell populations obtained from ipsilateral and contralateral SmLN of unilaterally SCGx mice. SmLN of mice parasympathetically decentralized by unilateral sections of the chorda tympani-lingual nerve trunk showed lower PFC response than the innervated contralateral SmLN, when challenged with SRBC 8-28 days after surgery. These results suggest a regulatory function of the autonomic nervous system in immune reaction.

Assuntos

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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.

Assuntos

RESUMO

The effect of local sympathetic denervation on immune responses of submaxillary lymph nodes (SmLN) of mice was examined in animals subjected to unilateral superior cervical ganglionectomy (SCGx). Norepinephrine (NE) content in ipsilateral SmLN decreased by 90

7-20 days after SCGx, while bilateral SCGx resulted in a 91

decrease of SmLN NE content. SmLN of mice subjected to unilateral SCGx exhibited greater plaque-forming cell (PFC) response than the innervated contralateral SmLN, when challenged i.d. or i.p. with sheep red blood cells (SRBC) 10-20 days after surgery. In mice challenged with SRBC at an early phase of sympathetic nerve paralysis (i.e., 2 h after SCGx), PFC response was already increased in the ipsilateral SmLN whereas during the anterograde degeneration of nerve endings (6-24 h after SCGx) an impending depression of PFC number was observed. Contact hypersensitivity and allogeneic delayed-type reactions were also enhanced in the ear ipsilateral to SCGx. Primed SmLN cells of nodes located ipsilaterally to SCGx, when injected to (BALB/c x C57BL/6) F1, resulted in significantly higher spleen index than similarly treated, contralateral SmLN. In contrast, mitogenic stimulation under various experimental protocols failed to reveal significant differences between proliferation of cell populations obtained from ipsilateral and contralateral SmLN of unilaterally SCGx mice. SmLN of mice parasympathetically decentralized by unilateral sections of the chorda tympani-lingual nerve trunk showed lower PFC response than the innervated contralateral SmLN, when challenged with SRBC 8-28 days after surgery. These results suggest a regulatory function of the autonomic nervous system in immune reaction.