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S L Lightman

Publications and source records attributed to S L Lightman.

At least 109 records · Page 6Linked to original sources
PubMed1997

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection.

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.

Animals↗
PubMed1997

Mitogenic response and steroid sensitivity in MS lymphocytes.

OBJECTIVES: We investigated mitogenic response and steroid sensitivity in multiple sclerosis (MS) lymphocytes to establish if MS lymphocytes were less steroid responsive. MATERIAL AND METHODS: We compared mitogenic response to phytohaemagglutinin (PHA) and inhibition by dexamethasone (DEX) in circulating lymphocytes from both MS patients and healthy subjects. RESULTS: We found a range of responses in each group but no significant differences between the two groups, nor in patients with and without enhancement on magnetic resonance imaging. The mid-inhibitory concentration of DEX in response to 1 microg/ml PHA was significantly lower than that for 2.5 microg/ml PHA in the patients. The mid-inhibitory concentrations of DEX in response to 2.5 microg/ml PHA negatively correlated with endogenous serum cortisol concentrations. CONCLUSION: These data imply a spectrum of glucocorticoid response that is similar in normal and MS lymphocytes and can partly explain why response to steroid therapy is variable.

Adult↗
PubMed1997

Emergence of an isolated arginine vasopressin (AVP) response to stress after repeated restraint: a study of both AVP and corticotropin-releasing hormone messenger ribonucleic acid (RNA) and heteronuclear RNA.

The hypothalamic-pituitary-adrenal axis adapts to periods of chronic or repeated stress. We have now investigated whether there is any differential effect of repeated immobilization stress on CRH and arginine vasopressin (AVP) gene transcription in the hypothalamic paraventricular nucleus (PVN), using probes for both intronic and exonic sequences of the AVP and CRH genes. Daily restraint for 13 days had no significant affect on basal corticosterone level, CRH heteronuclear RNA (hnRNA), CRH messenger RNA (mRNA), AVP hnRNA, or AVP mRNA. After the final episode of acute restraint, on day 14, there was a rapid increase in plasma corticosterone and AVP hnRNA level in the parvocellular subdivision of the PVN, a slower increase in AVP mRNA level in the parvocellular subdivision of the PVN, but no significant change in either CRH hnRNA or CRH mRNA in the PVN. The increase in AVP hnRNA and mRNA was specific to the parvocellular PVN, because there was no change in either transcript in the magnocellular cells of this nucleus. This isolated response is in marked contrast to naive rats, which show a predominant CRH hnRNA and mRNA response, and infers that part of the adaptation to repeated stress results from a down-regulation of CRH gene responsiveness with maintenance or even a compensatory increase in the AVP gene response.

Animals↗
PubMed1997

Central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats.

Endocrine responses to noise stress and anxiety-related behaviors were measured in groups of ovariectomized, estradiol-treated female rats given central infusions of oxytocin. Control animals receiving isotonic saline showed a large increase in plasma corticosterone concentrations in response to 10 min of white noise. This response to noise stress was significantly and dose dependently decreased by oxytocin administered intracerebroventricularly at 10 or 100 ng/h for 5 days. Oxytocin also significantly decreased rearing behavior during this stress. When a second noise stress was given 3 days after cessation of oxytocin infusion, corticosterone responses did not differ between the control and previously oxytocin-infused animals. Administration of vasopressin had no significant effect on either the corticosterone or behavioral responses to noise stress. Anxiety-related behaviors were measured on the elevated plus-maze. No significant differences were seen in maze exploration between saline- and oxytocin-treated animals when housed and tested in the same environment. However, when animals were mildly stressed by testing in an unfamiliar environment, oxytocin-treated animals showed a higher proportion of open arm entries and spent significantly more time in the open arms of the maze. Thus, oxytocin exerts a central anxiolytic-like effect on both endocrine and behavioral systems and could play a role in moderating behavioral and physiological responses to stress.

Animals↗
PubMed1997

Reduced response of the hypothalamo-pituitary-adrenal axis to alpha1-agonist stimulation during lactation.

To determine whether altered noradrenergic activation of the hypothalamo-pituitary-adrenal (HPA) axis contributes to the attenuated neuroendocrine response to stress observed during lactation, the effect of intracerebroventricular injection of the alpha1-agonist methoxamine (100 microg) was compared between virgin and lactating rats. Virgin rats showed significant increases in plasma corticosterone after methoxamine, reaching 317 +/- 44 ng/ml at 10 min and remaining significantly elevated for more than 120 min, but lactating rats showed no significant increase in corticosterone levels. Furthermore, methoxamine induced an increase in paraventricular nucleus (PVN) CRF messenger RNA expression in virgin, but not lactating, animals. Both groups of rats exhibited comparable elevations in plasma PRL after methoxamine treatment. Arginine vasopressin messenger RNA expression within the parvocellular PVN was greater in the lactating animals than in the virgin controls, but methoxamine injection was without further effect. Studies performed on ovariectomized virgin rats and ovariectomized rats receiving estradiol or progesterone replacement failed to reproduce the attenuated HPA responses seen after methoxamine treatment, although methoxamine-induced PRL levels were greatly increased by estradiol, probably arising from an effect on hormone synthesis. In vitro electrophysiological recordings of PVN neurons in hypothalamic slices from proestrous virgin and lactating rats showed that 45-52% of neurons in both groups exhibited excitatory responses to 10(-4) M methoxamine, but there was a differential response to 10(-5) M methoxamine, with PVN neurons from lactating animals failing to show a response. These data show a selective down-regulation of alpha1-mediated activation of the HPA axis in lactating animals. This may contribute to the attenuated stress-induced activation of the HPA axis during lactation.

Adrenergic alpha-Agonists↗
PubMed1997

Rapid changes in heteronuclear RNA for corticotrophin-releasing hormone and arginine vasopressin in response to acute stress.

The rapid detection of gene activation is important for our understanding of gene regulation. We have therefore studied heteronuclear (i.e. nascent) RNA (hnRNA) by using 35S-labelled corticotrophin-releasing hormone (CRH) riboprobes and arginine vasopressin (AVP) oligo-nucleotide probes directed against intronic and exonic sequences of both CRH and AVP transcripts for in situ hybridization studies of transcriptional changes during acute stress. CRH and AVP intronic signals (found in newly synthesized transcripts) were confined to the nuclei of the parvocellular cells in the paraventricular nucleus (PVN) whilst CRH and AVP exonic signals (found in both newly formed and mature transcripts) were primarily located in the cytoplasm of these cells. AVP hnRNA and mRNA were also present at high level, in the magnocellular PVN. The levels of CRH hnRNA and parvocellular AVP hnRNA in the PVN were significantly increased 1 and 2 h after the onset of restraint. The levels of CRH mRNA on the other hand were not significantly increased until 4 h after the onset of restraint. The number of AVP mRNA-expressing neurons in the medial parvo-cellular cells of the PVN significantly increased at 2 h and peaked at 4 h after the onset of stress. In contrast, densitometric analysis indicated that the increase in AVP mRNA levels in these cells did nor reach significant difference from control until 4 h after the onset of restraint. There were no significant changes on AVP hnRNA or AVP mRNA levels in the magnocellular subdivision of PVN at any time point after the onset of restraint.

Acute Disease↗
PubMed1997

Effects of maternal iodine deficiency and thyroidectomy on basal neuroendocrine function in rat pups.

We have used in situ hybridization histochemistry to investigate the effects of maternal thyroidectomy and chronic maternal iodine deficiency on basal neuroendocrine function in rat pups. Specifically, we have measured hypothalamic thyrotrophin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) expression together with circulating levels of tri-iodothyronine (T3) in rat pups delivered from and suckled by thyroidectomized or iodine-deficient dams. Because of the close interaction between the thyroid, adrenal and growth hormone axes, we have also examined hypothalamic corticotrophin-releasing hormone (CRH) and growth hormone-releasing hormone (GRH) transcripts at the same time points: birth, 1 month and 2 months of age. Three weeks after surgical thyroidectomy, adult female Sprague-Dawley rats proved unable to carry pups to term and lactate successfully. Pups delivered from thyroidectomized dams given a small replacement dose of T3 during pregnancy were significantly lighter than controls (84 +/- 3%) and had markedly depressed plasma T3 levels (36 +/- 6% of control). Hypothalamic CRH and GRH transcript levels were significantly decreased in pups at birth (to 8 +/- 2.5% and 24 +/- 8% of control respectively) but had returned to normal by 1 month after delivery. Pituitary TSH transcript levels and hypothalamic levels of TRH transcripts, however, were similar to those of controls. Only one of seven dams fed a low-iodine diet for 6 months produced live pups, and these were too few in number to produce significant data. Dams fed a low-iodine diet from 4 months before mating, however, did produce live pups and although they were not significantly lighter than control pups at birth, by 1 month after birth, they were significantly lighter (72 +/- 3% of controls). Circulating T3 levels were not significantly different from control at any time point examined. Hypothalamic TRH levels were significantly elevated at birth (451 +/- 138% of control), but this difference was not maintained at 1 or 2 months after birth despite the lactating dams being maintained on the low-iodine diet. Pituitary TSH levels showed an upward trend at all time points that reached significance at 1 month after birth (204 +/- 19%; P < 0.05). Hypothalamic CRH and GRH transcript levels were not different from controls at any time point. In summary, chronic iodine deficiency or thyroidectomy with low-level T3 replacement in Sprague-Dawley rats markedly impaired fertility and the ability to carry pups to term, and produced an unexpectedly modest up-regulation of the hypothalamo-pituitary-thyroid axis and down-regulation of the hypothalamo-pituitary-adrenal axis.

Animals↗
PubMed1997

Differential regulation of hypothalamic pituitary corticotropin releasing hormone receptors during development of adjuvant-induced arthritis in the rat.

The expression of corticotropin releasing hormone (CRH) in the hypothalamic paraventricular nucleus (PVN) and CRH receptor mRNA in the PVN and anterior pituitary was studied during development of adjuvant-induced arthritis in Piebald-Viral-Glaxo rats, using in situ hybridization techniques. As previously shown with i.p. hypertonic saline injection, basal and immobilization stress-stimulated CRH mRNA levels in the PVN were significantly lower than in controls 14 days after adjuvant injection. However, 7 days after injection, preceding the onset of inflammation, the increase of CRH mRNA following immobilization was significantly higher than in control rats. In contrast to other chronic stress paradigms, inflammation stress failed to induce type-1 CRH receptor (CRH-R1) mRNA in the PVN, either at 7 days, or at 14 days after adjuvant injection, when inflammation is present. The ability of acute immobilization to induce CRH-R1 mRNA in the PVN was not affected 14 days after adjuvant injection but parallel to the CRH peptide mRNA response it was markedly potentiated at 7 days. Pro-opiomelanocorpin (POMC) mRNA levels in the anterior pituitary increased significantly 14 days after adjuvant injection, and they were unaffected by 1 h immobilization. While CRH binding in the pituitary decreased significantly 14 days after adjuvant injection, CRH-R1 mRNA was unchanged. This study shows biphasic hypothalamic responses to acute stress during development of adjuvant-induced arthritis, with enhanced CRH peptide and CRH-R1 mRNAs responses at 7 days, preceding the onset of inflammation, and blunted CRH mRNA responses at 14 days at the height of the inflammatory response. The lack of CRH receptor expression in the PVN in this model of chronic inflammation stress associated to low hypothalamic CRH peptide levels supports the view that positive feedback regulation by CRH is necessary to maintain enhanced CRH expression during chronic stress.

Animals↗
PubMed1997

Interleukin-1 beta-induced disruption of the retinal vascular barrier of the central nervous system is mediated through leukocyte recruitment and histamine.

The vascular barriers of the central nervous system form a selective cellular interface between the blood and the neural parenchyma and restrict the transfer of both molecules and hematogenous cells. During immune-mediated diseases, leukocyte infiltration becomes dramatically up-regulated and the permeability of these barriers increases, leading to edema formation. The etiology of this damage remains largely unresolved although inflammatory cytokines have been implicated in the process. The effect of the proinflammatory cytokine interleukin (IL)-1 beta on the integrity of the rat blood-retinal barrier (BRB) was investigated up to 14 days after an intravitreal injection. The permeability of the BRB was evaluated quantitatively using the low molecular weight tracer [14C]mannitol. After IL-1 beta administration, a biphasic opening of the BRB to [14C]mannitol was recorded, peaking at 4 to 8 hours and 24 to 48 hours post-injection (PI). The early disruption coincided with the appearance of both polymorphonuclear and mononuclear leukocytes within the retina. By 12 hours PI, BRB permeability had returned to control values despite a continued increase in the number of infiltrating leukocytes. The second, more pronounced increase in barrier permeability detected at 24 to 48 hours PI corresponded with maximal leukocyte infiltration. Barrier dysfunction had resolved by 72 hours, and by 7 days the leukocyte infiltrate had disappeared. The IL-1 beta-induced increase in permeability could be completely abrogated at 4 and 24 hours PI by treating the animals with the histamine H2-receptor antagonist ranitidine, which also reduced leukocyte infiltration by 47.2%. The ability of histamine to disrupt the BRB was demonstrated by intravitreal and intravascular administration, which caused a rapid and significant increase in BRB permeability. Treatment of the animals with the cyclo-oxygenase inhibitor indomethacin had no effect on IL-1 beta-induced disruption of the BRB at 4 hours PI, but by 24 hours PI a significant reduction in permeability was observed that coincided with a 75.2% reduction in the leukocyte infiltrate. The depletion of circulating leukocytes to < 2% of control levels reduced the retinal leukocyte recruitment induced by IL-1 beta by 73.0% and decreased BRB permeability at both 4 and 24 hours after IL-1 beta injection. These data demonstrate that intravitreal IL-1 beta in the rat induces a biphasic opening of the BRB that appears to be mediated through recruited leukocytes and release of the vasoactive amine histamine.

Animals↗
PubMed1997

Ultrastructural analysis of interleukin-1 beta-induced leukocyte recruitment to the rat retina.

PURPOSE: To examine, by ultrastructural analysis, the effect of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) on the integrity of the rat retina and blood-retinal barrier (BRB) and to investigate the site of barrier leakage and the path of leukocyte infiltration into the retina. METHODS: After a single injection of IL-1 beta into the vitreous of the Lewis rat, leukocyte recruitment and retinal disease was assessed immediately and at frequent periods up to 14 days after injection by light and electron microscopy and by immunohistochemistry. The integrity of the BRB and the site of barrier disruption were evaluated using the large molecular weight tracer horseradish peroxidase. The phenotype of recruited leukocytes to the retina was assessed by ultrastructural morphology and immunohistochemistry. RESULTS: At 4 hours after the intravitreal administration of IL-1 beta, leukocytes were observed infiltrating the retina. Leukocyte infiltration increased gradually and peaked between 24 and 48 hours after injection. Associated with this infiltrate were edema and fibrin leakage, indicative of a breakdown in the BRB. This was confirmed by the demonstration of horseradish peroxidase extravasation across the retinal vascular bed, with leakage of the tracer through disrupted endothelial tight junctions. Using immunohistochemical and morphologic criteria, the majority of infiltrating cells were identified as monocytes-macrophages and neutrophils; occasionally, T cells were found. Ultrastructural analysis showed that the majority of cells entered the retina by migrating through retinal endothelial cells and that there was a smaller contribution from the ciliary body. CONCLUSIONS: The administration of IL-1 beta to the vitreous of the Lewis rat causes an acute, reversible retinal inflammatory response that is accompanied by breakdown of the vascular BRB. Interleukin-1 beta induces the recruitment of mononuclear and polymorphonuclear leukocytes that enter the retina predominantly through the retinal vasculature and that appear to migrate through retinal endothelial cells. These results suggest that IL-1 beta may be an important factor in the pathogenesis of human retinal inflammation.

Animals↗
PubMed1996

Region-specific reduction in stress-induced c-fos mRNA expression during pregnancy and lactation.

Hypothalamo-pituitary-adrenal (HPA) responses to stress are dramatically attenuated during lactation. To examine whether this is due to diminished stress-induced activation of specific areas of the brain involved in HPA responses, c-fos mRNA expression was employed as a marker of stress-induced neuronal activation. Regional levels of expression were quantified in female rats exposed to 30 min immobilisation stress during late pregnancy (days 19-21), early lactation (days 3-4) and mid-lactation (days 10-14), and compared with the levels in virgin females. Stress-induced levels of corticosterone were significantly lower in late pregnant and early lactating rats compared with the levels in virgin females, and this correlated with a marked attenuation of stress-induced c-fos mRNA expression in the parvocellular division of the PVN. This reduced activation suggests that neuroendocrine hyporesponsiveness during lactation may arise from an effect on afferent pathways to the PVN. Extrahypothalamic areas known to be important for HPA activation displayed three patterns of c-fos mRNA expression: (i) in the ventral tegmental area, dorsal vagal complex, pyriform cortex and all areas of the hippocampus (CA1, CA2, CA3, dentate gyrus), expression levels did not vary significantly with reproductive status; (ii) in the locus coeruleus (A6 catecholaminergic group), a peak of expression was detected in late pregnant animals; and (iii) in the medial amygdala, ventral part of the lateral septum and cingulate cortex expression was significantly reduced in pregnant and lactating animals, with a nadir in early lactation. The decreased expression of c-fos mRNA in these latter areas correlated with that in the parvocellular PVN, and suggests that their interaction may contribute to the reduced neuroendocrine responses of lactating rats.

Animals↗
PubMed1996

Phenotypic characterization of T cells infiltrating the conjunctiva in chronic allergic eye disease.

BACKGROUND: Chronic allergic conjunctivitis comprises a spectrum of diseases including atopic keratoconjunctivitis, atopic blepharoconjunctivitis, vernal keratoconjunctivitis, and contact lens-associated giant pupillary conjunctivitis. Each condition is characterized by a complex immunopathology with a mixed cellular infiltrate. Treatment with conventional mast cell stabilizers is often unsatisfactory, and therapy depends heavily on topical corticosteroids. OBJECTIVE: Recent evidence suggests that T lymphocytes play an important role in mediating the immunopathology seen in the chronic allergic response in the skin and the lungs. METHODS: We have therefore investigated the prevalence of T cells and their subsets in the different chronic allergic eye conditions by means of immunohistochemistry. RESULTS: We found significantly increased numbers of CD4+, CD45RO+, and HLA-DR+ T cells in the conjunctiva of patients with atopic and vernal keratoconjunctivitis and giant papillary conjunctivitis, with a corresponding upregulation of markers present on antigen presenting cells. CONCLUSIONS: These findings suggest that like allergic conditions in the skin and lungs, CD4+ memory T cells are involved in the regulation of the immunopathology of chronic allergic eye responses. Further characterization of these T cells will provide the information necessary for future immunotherapeutic interventions.

Adolescent↗
PubMed1996

Role of LFA-1, ICAM-1, VLA-4 and VCAM-1 in lymphocyte migration across retinal pigment epithelial monolayers in vitro.

The blood-retinal barrier (BRB), which is composed of the retinal pigment epithelium (RPE) and retinal vascular endothelium, normally restricts the traffic of lymphocytes into the retina. During ocular inflammatory conditions such as posterior uveitis there is a large increase in lymphocyte migration across the BRB. The differential role played by the two barrier sites, however, remains unclear. To evaluate the role of the posterior BRB, the migration of CD4+ antigen-specific T-cell line through rat RPE cell monolayers was investigated in vitro using time-lapse videomicroscopy. The adhesion molecules involved in controlling transepithelial migration across normal and interferon-gamma (IFN-gamma)-activated RPE was assessed with monoclonal antibodies directed against cell adhesion molecules. Lymphocytes were treated with antibodies specific for CD11a (alpha L subunit of LFA-1), CD18 (beta 2 subnit of the leucam family) and CD49 d (alpha 4 subnit of very late activation antigen-4, VLA-4), and the RPE with antibodies specific for CD54 (intracellular adhesion molecule-1, ICAM-1) and CD 106 (vascular cell adhesion molecule-1, VCAM-1). Migration across unstimulated RPE was inhibited by antibodies to ICAM-1 (48.6 +/- 3.5% reduction), leucocyte functional antigen-1 (LFA-1) alpha (61 +/- 5.2%) and LFA-1 beta (63.2 +/- 4.7%), but not by antibodies to VLA-4. VCAM-1 was not expressed on untreated RPE. Following activation of the RPE monolayers for 72 hr with IFN-gamma, antibodies to LFA-1 alpha, LFA-1 beta and ICAM-1 inhibited migration by 49.9 +/- 9.4%, 63.6 +/- 5.5% and 47.7 +/- 4.2% respectively. Antibodies to VLA-4 and VCAM-1 blocked migration by 21.5 +/- 8.4% and 32.3 +/- 6.2%, respectively, which correlated with the induction of VCAM-1 expression on RPE and increased migration. Under these conditions blocking both VCAM-1 and ICAM-1 reduced migration by 70.9 +/- 2.3%, which was greater than the effect of blocking either of these molecules alone. These results demonstrate that the posterior barrier of the BRB utilizes the same principle receptor-ligand pairings in controlling lymphocyte traffic into the retina as the vascular endothelium of the anterior BRB.

Animals↗
PubMed1996

The role of endogenous serotonin in adjuvant-induced arthritis in the rat.

We have investigated the effects of serotonin depletion on the progress and severity of adjuvant-induced arthritis in the Piebald-Viral-Glaxo (PVG) strain of rat. Total body depletion of serotonin was achieved using p-chlorophenylalanine given i.p. Two paradigms were investigated. First we depleted serotonin at the time of injection of the adjuvant to determine whether serotonin was involved in the initial induction phase. Secondly, we depleted serotonin at the time of onset of the inflammation. Serotonin levels in the hypothalamic paraventricular nucleus (PVN) were reduced by > 95%. Depletion at the time of induction had no effect on the severity of the disease (determined by the increase (determined by the increase in hind paw volume) 14 days after injection of the adjuvant. In contrast, depletion at the time of onset of the disease resulted in a significant reduction in severity at day 14, suggesting a pro-inflammatory role for serotonin in this model. The decrease in corticotrophin-releasing factor (CRF) mRNA in the PVN associated with the development of adjuvant arthritis in PVG rat was reversed in the serotonin-depleted animals. Central serotonin could be one of the factors responsible for the reduced expression of CRF mRNA in response to adjuvant-induced arthritis in this rat strain. These data suggest that serotonin antagonists may be efficacious in reducing the severity of acute inflammatory episodes.

Animals↗
PubMed1996

Effects of extracellular nucleotides in the pituitary: adenosine triphosphate receptor-mediated intracellular responses in gonadotrope-derived alpha T3-1 cells.

We have recently identified gonadotropes as target cells for ATP action via ATP receptors of the P2U subtype. The present studies have used gonadotrope-derived alpha T3-1 cells to examine the possible signaling mechanisms subserving ATP action in gonadotropes. Addition of ATP produced a biphasic intracellular Ca2+ (Ca2+i) response: a transient spike followed by a small plateau. Removal of extracellular Ca2+ or depolarization with KCl abolished the plateau but had no effect on the spike. The plateau was also blocked by cadmium or nifedipine but not nickel. Pretreatment with GnRH or thapsigargin but not ryanodine inhibited the subsequent Ca2+i response to ATP. Pertussis toxin had no effect on ATP-induced Ca2+i response, whereas the phospholipase C inhibitor U73122 reduced the response. These observations suggest that the Ca2+i response is mediated by a pertussis toxin-insensitive and phospholipase C-coupled G-protein and reflects Ca2+ release from the GnRH- and thapsigargin-sensitive Ca2+ pool followed by Ca2+ influx through high voltage-gated Ca2+ channels. Activation of these ATP receptors had no apparent effects on the cAMP and cGMP signaling systems. Treatment with ATP-gamma S caused the translocation of protein kinase C (PKC) epsilon but not PKC zeta and PKC alpha to the particulate fraction. These data not only characterize the ATP receptor-mediated intracellular signaling in alpha T3-1 cells and render further evidence for a mediator role for nucleotides in gonadotrope function but also provide the first direct demonstration of PKC translocation by ATP receptors.

Biological Transport↗
PubMed1996

Persistent transgene expression in the hypothalamus following stereotaxic delivery of a recombinant adenovirus: suppression of the immune response with cyclosporin.

Replication deficient, recombinant adenoviruses (Ads) have been used successfully to transfect several forebrain and brainstem nuclei, but have yet to be demonstrated as useful vectors for transgene delivery in the structurally diverse and highly vascularised nuclei of the hypothalamus. In the present study we have assessed the ability of an Ad expressing the lac-Z gene to transfect cells of the paraventricular nucleus (PVN) of the hypothalamus in vivo. We show that: (1) we can achieve stable expression of the lacZ gene in cells of the magnocellular PVN for at least 2 months; (2) there were no obvious differences in the level of AVP mRNA in the PVNs injected with Ad compared with those injected with vehicle suggesting that Ad treatment is not disrupting normal cellular function in the injection region; (3) the introduction of Ads results in a limited immune response; (4) systemic treatment with cyclosporin dramatically reduces its magnitude. We conclude that Ad vectors represent useful tools for neuroendocrinological and gene therapeutic studies of the hypothalamus.

Adenoviruses, Human↗
PubMed1996

Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor.

There is increasing evidence that pituitary ATP receptors may play a novel role in modulating pituitary function. This work reports the isolation and expression of a pituitary ATP receptor gene clone from a rat pituitary complementary DNA library. The isolated clone (rpP2U) has a 1125-bp coding sequence flanked by 483 bp of 5' - and 422 bp of 3'-untranslated sequences. The deduced 374-amino acid product shows structural features common to other G protein-coupled receptors, and when stably transfected into a glioma cell line lacking endogenous ATP receptors, is functionally characterized as a P2U purinoceptor. Specifically, the ATP-induced intracellular Ca2+ mobilization in the transfected cells was inhibited by suramin, 2-methylthio-ATP had a modest stimulatory effect on intracellular Ca2+ mobilization, and beta, gamma-methylene ATP and alpha, beta-methylene ATP had no effect. The cloned receptor exhibited the agonist potency and efficacy profile of ATP approximately equal to uridine triphosphate > ADP approximately equal to uridine diphosphate > GTP. Such characteristics very closely mimic the pharmacologically defined P2U purinoceptor of primary rat gonadotropes and mixed sheep pituitary cells, and Southern blot analysis further indicates that there is only one allele in rat genome for the P2U purinoceptor. These findings suggest that the P2U purinoceptor is the predominant G protein-linked ATP receptor found in the pituitary.

Adenosine Triphosphate↗