Stromal cell-derived factor 1-alpha (SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-theta.

Stromal cell-derived factor 1alpha (SDF-1alpha) is the exclusive ligand for the chemokine receptor CXCR4. This receptor plays a pivotal role in immune responses, the pathogenesis of infection such as HIV, and cellular trafficking. However, the signaling mechanisms regulating SDF-driven T cell migration are not well defined. In this study, we determined the role of PI3K and protein kinase C- theta (PKC-theta) in SDF-induced human T cell migration in fresh versus cultured T cells. Purified human T cells (fresh vs. 48 h in media, unstimulated or activated by anti-CD3+anti-CD28) were used. Western blots showed that SDF induced phospho-(p)-Akt [threonine (Thr)308 and serine 473], a proxy for PI3K activity, in fresh cells and p-PKC-theta in 48 h unstimulated cells. LY294002 (PI3K inhibitor) reduced SDF-induced chemotaxis in fresh cells by 51%, whereas it minimally affected chemotaxis in 48 h unstimulated or activated cells. However, a specific PKC-theta inhibitor, pseudosubstrate for PKC-theta, reduced chemotaxis in 48 h unstimulated and stimulated T cells by 72% and 87%, respectively. Thus, chemotaxis becomes independent of PI3K signaling in human T cells cultured for 48 h. Under these conditions, PKC-theta is phosphorylated (Thr538) by SDF, and chemotaxis becomes largely PKC-theta-dependent.

Recombinant factor VIIa for the treatment of severe postoperative and traumatic hemorrhage.

BACKGROUND: The aim of this study was to determine the dose of recombinant factor VIIa (rFVIIa) that has been used in our institution to successfully control hemorrhage in trauma and postoperative patients. METHODS: This was an 8-month retrospective cohort study of 13 patients with acute hemorrhage and no known history of coagulopathic disorders. RESULTS: Administration of factor VIIa resulted in the cessation of life-threatening hemorrhage at dosages approximately one half those recommended for the management of hemophilia. After administration, there was a significant decrease in the total blood-product transfusion requirement (P <0.05). CONCLUSIONS: The use of factor VIIa in patients with life-threatening hemorrhage is a safe and effective therapeutic modality when used as an adjunct to standard interventions for control of severe hemorrhage. Lower-dose regimens were as successful as higher-dose regimens previously reported. The results of this respective study of 13 patients suggests that recombinant factor VIIa therapy for control of life-threatening hemorrhage as an adjunct to standard interventions can be successful at doses <90 mg/kg.

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT-PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes froin CD 1 female mice as well. The results of studies using quantitative competitive RT-PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti-CD3-epsilon for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti-CD3-epsilon did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti-CD3-epsilon. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti-CD3-epsilon-stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti-CD3-epsilon regulates DOR expression differently than PMA and ionomycin.

Tumor necrosis factor-alpha is a potent ACTH secretagogue: comparison to interleukin-1 beta.

Tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) are secreted by activated monocytes and other immune cells. Since IL-1 has been shown to elevate rat plasma ACTH and both of these cytokines induce similar acute-phase responses, the present studies of TNF were undertaken to characterize the ACTH response to this immune cell product. Human rTNF, administered iv at doses (100-1000 ng) which failed to affect blood pressure, food consumption or prolactin levels, resulted in significant peak elevations of rat plasma ACTH within 20 min (mean +/- SE 304 +/- 94 and 958 +/- 128 pg/ml for 100 and 1000 ng, respectively, compared to 53 +/- 16 pg/ml for vehicle). rTNF from two different sources produced similar elevations of ACTH as an equivalent amount of rIL-1. TNF failed to affect cultured anterior pituicytes, and it did not modify the response to CRF. When administered into the upper third cerebroventricle, TNF 20 ng failed to affect ACTH levels whereas IL-1 30 ng raised ACTH to 638 +/- 79 pg/ml compared to 177 +/- 24 pg/ml for vehicle (p less than .001). Furthermore, intraparenchymal injection of IL-1, directly above the median eminence, elevated ACTH to 484 +/- 93 pg/ml; again, TNF was completely ineffective. Thus, TNF-alpha and IL-1 beta are both potent ACTH secretagogues with complementary modes of action; however, the proximate target of TNF action appears to be peripheral to the CNS and pituitary whereas that of IL-1 appears to be the median eminence.

Delta opioid receptors expressed by stably transfected jurkat cells signal through the map kinase pathway in a ras-independent manner.

Delta opioid receptors (DOR) are G-protein coupled 7-transmembrane receptors (GPCR), expressed by thymic and splenic T cells, that modulate interleukin (IL)-2 production and proliferation in response to concanavalin A or crosslinking the TCR. Mitogen-activated protein kinases (MAPKs) are involved in mediating intracellular responses to TCR crosslinking. In addition, MAPKs can be activated by signaling cascades that are initiated by the release of G-proteins from GPCRs. To determine whether DORs expressed by T cells signal through the MAPKs, extracellular-regulated kinases (ERKs) 1 and 2, two delta opioid peptides, deltorphin and [D-Ala2,D-Leu5]-enkephalin (DADLE), were studied in Jurkat cells that had been stably transfected with DOR (DOR-Ju.1). These peptides rapidly and dose-dependently induced ERK phosphorylation; pretreatment with naltrindole (NTI), a selective DOR antagonist, abolished this. Pertussis toxin (PTX) also inhibited phosphorylation, indicating the involvement of the Gi/o proteins. Herbimycin A, a protein tyrosine kinase (PTK) inhibitor, reduced the DADLE-induced ERK phosphorylation by 68%. ERK phosphorylation was inhibited by Bisindolylmaleimide 1 (GF109203X), an inhibitor of PKC, and by pretreatment with PMA prior to DADLE. A GTP/GDP exchange assay was used to assess the potential role of Ras in the pathway leading to ERK phosphorylation; DADLE failed to stimulate GTP/GDP exchange in comparison to PMA. Additional studies showed that DADLE stimulated an increase in cfos mRNA; this was reduced by the inhibitor of MAPK/ERK kinase (MEK), PD98059. Therefore, in DOR-Ju.1 cells, DOR agonists stimulate ERK phosphorylation in a Ras independent and PKC-dependent manner; PTKs appear to be involved. MAPKs mediate the increase in cfos mRNA induced by DOR agonists.

Detection of basal levels and induction of delta opioid receptor mRNA in murine splenocytes.

Activation of delta opioid receptors (DOR) modulates calcium mobilization, interleukin-2 production, chemotaxis and proliferation of T-lymphocytes. Recent reports indicate that lymphocytes and mononuclear cells may express mRNA transcripts for DOR. The investigations reported herein show that low levels of DOR were consistently detected by RT-PCR amplification of RNA from freshly obtained Balb/c murine splenocytes, both weanling and adult. Culturing cells without stimulation increased DOR levels and concanavalin A apparently reduced this; DOR was preferentially expressed in a T-cell-enriched fraction. Thus, the expression of DOR mRNA by unactivated splenocytes is modulated by culture and con A in the T-cell fraction.

Signaling through delta opioid receptors on murine splenic T cells and stably transfected Jurkat cells.

beta-Endorphin (beta-EP) and delta opioid receptor (DOR) agonists affect immune functions such as lymphocyte chemotaxis, proliferation, and cytokine production. Recent studies indicate that both neuronal DOR and novel G-protein-coupled receptors with high affinity for beta-EP and DOR agonists are expressed by mononuclear cells. In addition, proenkephalin A mRNA and enkephalin-related peptides are expressed by lymphocytes. These investigations were conducted to identify signal transduction pathways that mediate the effects of beta-EP and DOR agonists on T cells. Calcium mobilization was studied because it is central to T-cell activation initiated by antigen presentation to the T-cell receptor (TCR). Using the calcium-sensitive dye Fluo-3 and flow cytofluorometry to determine the concentration of free intracellular calcium, physiological concentrations of beta-EP were shown to enhance concanvalin. A (con A)-stimulated calcium mobilization by murine splenic T cells (p < 0.01). The DOR antagonist, naltrindole, inhibited this, whereas CTAP, a selective mu OR antagonist, was ineffective. In addition, N-Ac-beta-EP and the mu OR agonist DAMGO, failed to mimic the effects of beta-EP. Although it was less potent than beta-EP, DADLE, a DOR agonist, also enhanced Con-A-induced calcium mobilization (p < 0.01). A DOR-transfected human T-cell line (DOR-Jul.1) was developed to study signal transduction. Both DADLE and the selective DOR agonist, deltorphin, rapidly increased intracellular free calcium concentrations; ED50s were 10(-9) M. Pertussis toxin prevented the response, and EGTA significantly reduced it. In addition, DADLE inhibited forskolin-stimulated cAMP production (ED50: 10(-11) M). These findings with normal splenic T cells and DOR-transfected T-cell line indicate that beta-EP and DOR agonists affect calcium mobilization. This is likely to modulate downstream pathways that regulate T-cell activation and function.

Expression of delta opioid receptors and transcripts by splenic T cells.

Delta opioid receptors (DORs) and preproenkephalin-A-derived opiate peptides are expressed by mononuclear cells in various lymphoid organs. DOR ligands modulate a variety of immune functions, such as T-cell proliferation, calcium mobilization, and cytokine production. Recently, quiescent T cells were found to express low levels of DOR transcripts, which increased due to the following: cell culture of unstimulated murine splenocytes (depending on cell density); cross-linking the T-cell receptor (TCR) with anti-CD3-epsilon; and a single in vivo exposure to staphylococcal enterotoxin B (SEB). Enhanced expression of DOR mRNA was mediated transcriptionally. Moreover, PMA + ionomycin, which mimic the proliferative signal of anti-CD3, inhibited the expression of DOR mRNA. Using semiquantitative immunofluorescence to detect DORs, SEB was found to increase the fraction of T cells that expressed DOR and to enhance the relative level of DOR expression per T cell. Previous studies have shown that DOR agonists inhibited the anti-CD3-stimulated production of interleukin-2 and T-cell proliferation. Therefore, the enhanced expression of DORs by activated T cells may be capable of downregulating the T-cell activation program.

Nicotine administration enhances NPY expression in the rat hypothalamus.

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Reducing delays in the CT scanning process at Bloomington Hospital.

UNLABELLED: Over a two-month period in 1992, a team in the Radiology Department at Bloomington Hospital recorded the number and types of delays that occurred in the computer tomography (CT) scanning process. Using the Pareto principle, the team identified two root causes for delays--equipment failure and insufficient information collected at scheduling. Appropriate interventions were identified, and the number of delays decreased dramatically. RESULTS: The number of delays in the CT scanning process decreased from a median of 19 per day to 2. Staff overtime hours decreased from a median of 87 per month to 46. The time patients spend waiting to receive a CT scan decreased from an average of 20 minutes to 30 minutes to 0 to 10 minutes. Patients are scheduled for a scan within one day's notice, as opposed to one week previously. Complaints from patients, physician offices, and other customers decreased dramatically. Morale and empowerment improved among staff.

Thrombolytic therapy of acute ischemic stroke during pregnancy.

The authors report eight pregnant women with acute ischemic stroke treated with thrombolysis (rt-PA [recombinant human tissue plasminogen activator] or urokinase). Seven women recovered. Two extracranial and two asymptomatic intracranial hemorrhages complicated treatment; one woman died of arterial dissection complicating angiography. Three patients had therapeutic abortions, two fetuses were miscarried, and two babies were delivered healthy. Although pregnant women may be treated safely with thrombolytics, risks and benefits to mother and fetus must be carefully weighed.

Phosphorylation of activating transcription factor in murine splenocytes through delta opioid receptors.

Delta opioid receptors (DORs) modulate TCR signaling through the mitogen-activated protein kinases (MAPKs), ERKs 1 and 2. These studies determined whether a DOR agonist alone ([D-Ala(2)-D-Leu(5)]enkephalin; DADLE) affects phosphorylation of the activating transcription factor (ATF-2) and its interaction with the MAPK, c-Jun NH(2)-terminal kinase (JNK). DOR expression was induced on murine splenocytes by anti-CD3 and then quiescent cells were treated with DADLE. DADLE, itself, dose-dependently induced maximal phosphorylation of ATF-2 within 5-10min; naltrindole, a specific antagonist, abolished this. Anti-ATF-2 immunoprecipitates from control and DADLE-treated splenocytes showed a dominant 59kDa phosphorylated band and a 71kDa band. DADLE stimulated phosphorylation of both bands, although the 71kDa band was selectively immunoprecipitated by anti-JNK. Thus, DADLE stimulated phosphorylation of 71kDa ATF-2 and its association with JNK, suggesting that JNK is activated through DORs. Along with previous observations, these studies suggest that lymphocyte DORs can affect the activation of MAPKs by TCR-independent stimulation (e.g., JNK) or indirectly by modulating TCR-dependent stimulation (e.g., ERK).

Role of the fourth cerebroventricle in mediating rat plasma ACTH responses to intravenous nicotine.

Peripherally administered nicotine elevates rat plasma adrenocorticotropic hormone (ACTH) levels, acting at brain regions adjacent to or downstream from the third cerebroventricle. Studies evaluated whether the paraventricular nucleus (PVN) mediates directly the ACTH response to nicotine administered i.v. and if regions adjacent to the fourth ventricle (IV) are involved. Direct involvement of the PVN in the release of ACTH in response to i.v. nicotine (0.03 or 0.01 mg/kg b.wt.) was refuted by studies in which the administration of the nicotinic cholinergic antagonist, mecamylamine (20 or 40 micrograms bilaterally adjacent to the PVN), failed to block ACTH secretion. To activate sites distal to the third ventricle, nicotine (0.25, 0.5, 2.5 or 5.0 micrograms) was injected into the IV; ACTH levels peaked between 3 and 7 min. Nicotine 0.25 micrograms injected into the IV elevated ACTH to levels within the range of those produced by i.v. nicotine (0.03 mg/kg b.wt.). To confirm that sites accessible from the IV are involved, mecamylamine was administered i.v. (0.5 or 1.0 mg/kg b.wt.) or into the IV (4 or 40 micrograms) and nicotine was delivered by the opposite route. Intravenous mecamylamine reduced the plasma ACTH response to 0.25 micrograms of nicotine in the IV. Mecamylamine, administered into the IV before i.v. nicotine (0.03 mg/kg b.wt.) antagonized the effect of nicotine. These results indicate that stimulation of ACTH secretion by nicotine delivered i.v. occurs via structures accessible from the IV. In contrast to prevailing ideas, the PVN does not appear to be the direct site of action of nicotine.

Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase.

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 microg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 +/- 0.62 units (mean +/- SEM), increasing to 16.1 +/- 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 +/- 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10(-8) to 10(-6) M, [d-Ala(2)-d-Leu(5)]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.

Induction and Desensitization of the c-Fos mRNA Response to Nicotine in Rat Brain.

Early rapid response genes such as c-fos are activated in the central nervous system by a variety of agents including psychostimulants. In the present studies, we investigated changes in c-fos mRNA content in several brain regions of the rat in response to nicotine. A single injection of nicotine ip increased the c-fos mRNA content within 30 min and returned toward baseline by 120 min. Significant elevations were induced by 0.5 mg/kg bw nicotine in the medial habenula and by 1.0 mg/kg in the hippocampus, dentate gyrus, and piriform cortex. At 1.0 mg/kg, significantly greater increases in c-fos mRNA levels were present in medial habenula and hippocampus compared to dentate gyrus and in dentate gyrus compared to piriform cortex. Moreover, at 1.0 mg/kg nicotine, increases were significantly less in cerebellar cortex and cingulate gyrus, and these were not dose-dependent. Mecamylamine, a nicotinic cholinergic receptor antagonist, significantly attenuated or eliminated c-fos mRNA response to 1.5 mg/kg nicotine in all regions, except in the cerebellar cortex. Desensitization of the c-fos mRNA response to nicotine was investigated by administering two injections of 2.0 mg/kg nicotine 2 h apart. In the hippocampus, dentate gyrus, and piriform cortex, the first dose of nicotine significantly reduced the c-fos mRNA response to a second dose. The magnitude of desensitization ranged from 43% (piriform cortex) to 81% (hippocampus). In summary, nicotine rapidly elevated the c-fos mRNA content in several rat brain regions. The sensitivity of this response to nicotine and development of desensitization differed among the regions.

Nicotine elevates rat plasma ACTH by a central mechanism.

Nicotine is a potent secretagogue for the release of adrenocorticotropin (ACTH) from the anterior pituitary in vivo. However, the location of its action is unknown; knowledge of this is essential for elucidating its mechanism. Our studies show that cytisine, a peripherally acting nicotinic cholinergic agonist, given i.v. at doses equimolar or greater than nicotine, failed to elevate plasma ACTH levels, whereas nicotine (0.01 and 0.03 mg/kg b.wt.) had significant effects. Nicotine (10(-7)-10(-4) M) had no effect on the secretion of beta-endorphin by anterior pituicytes in vitro, nor did it potentiate the action of corticotropin-releasing factor (10(-9) or 10(-8) M). Intracerebroventricular injection of nicotine (1-20 micrograms) significantly elevated ACTH levels. Moreover, ACTH responses to nicotine delivered into the hypothalamic region of the third ventricle were significantly greater than those elicited by injection into the upper region. Additional studies were conducted to determine the earliest age at which nicotine stimulates ACTH. The response to i.p. nicotine (1 or 2 mg/kg b.wt.) was present but diminished during the postnatal period, whereas maximal responses comparable to mature rats were attained by day 15. To establish whether nicotine has a central effect in younger animals, nicotinic antagonists were tested. Hexamethonium (2 mg/kg b.wt.), a peripherally acting antagonist, was ineffective against nicotine (0.025 and 2.0 mg/kg b.wt.), whereas mecamylamine (2 mg/kg b.wt.), inhibitory at both peripheral and central sites, blocked the ACTH response. Thus, whether administered peripherally or centrally, nicotine activates central mechanisms mediating the release of ACTH; it appears that the target(s) for nicotine are within the hypothalamus or brainstem.

Immunofluorescence detection of delta opioid receptors (DOR) on human peripheral blood CD4+ T cells and DOR-dependent suppression of HIV-1 expression.

The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-([2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,-,(2S[1(S*),2alpha,5beta])-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 +/- 0.70 (mean +/- SEM) to 20.70 +/- 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed (approximately 50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

Attenuation of the plasma prolactin response to restraint stress after acute and chronic administration of nicotine to rats.

We have previously shown that a single dose of nicotine elevates plasma adrenocorticotropin (ACTH) levels in rats and has a biphasic effect on plasma prolactin (PRL). The stimulatory effect of nicotine on these stress responsive hormones desensitizes after a single injection of nicotine. Continuous exposure to nicotine also induces tolerance to its locomotor depressive and hypothermic effects, which have been associated with an increase of central [3H]nicotine binding. Thus, the acute and chronic administration of nicotine might induce changes in central nicotinic cholinergic circuits that affect the ACTH and PRL responses to stress. In the present study, a single dose of nicotine (0.75-3.0 mg/kg b.wt.) significantly inhibited the elevation of plasma PRL due to restraint stress initiated 60 min afterward. Five injections of nicotine during 1 day produced a similar attenuation of the PRL response to restraint stress but neither of these paradigms affected ACTH. In contrast, intermittent delivery of nicotine for 7 days failed to affect the PRL response to restraint stress; however, after withholding nicotine for 14 hr, high dose nicotine attenuated the PRL response to stress, whereas low dose nicotine remained ineffective. On the other hand, administration of the same schedule of low dose nicotine did significantly diminish the expected release of PRL in response to a final injection of nicotine (0.5-2.0 mg/kg b.wt.) in unstressed animals. In summary, a single dose or 5 doses of nicotine in 1 day attenuated the PRL response to restraint stress, whereas, after chronic administration, this effect was lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.