A non-acromegalic case of multiple endocrine neoplasia type 1 accompanied by a growth hormone-releasing hormone-producing pancreatic tumor.

Cases of acromegaly due to GHRHproducing pancreatic endocrine tumors have been reported. Here we present a case of a 31-yr-old nonacromegalic man with hyperparathyroidism and elevated serum IGF-I with normal serum GH levels. Serum GH was not suppressed below 1 ng/ml by the glucose tolerance test and increased in response to TR H and GHRH administration. Magnetic resonance imaging (MRI) revealed pituitary hyperplasia and an abdominal computed tomography (CT ) scan showed a tumor in the pancreatic tail. Plasma concentration of GHRH was elevated. Based on these clinical data, multiple endocrine neoplasia (MEN) type 1 was suspected. Three enlarged parathyroid glands were removed and a distal pancreatectomy was performed. Pathological examination of the parathyroid glands and pancreatic tumor showed nodular hyperplasia and a well-differentiated endocrine tumor, respectively, both compatible with MEN features. Immunohistochemistry revealed positive immunoreactivity for GHRH, SS , insulin, glucagon, chromogranin A, and pancreatic polypeptide in the pancreatic tumor. After pancreatic surgery, elevated levels of GHRH and IGF-I were normalized and pituitary hyperplasia definitely decreased in size. In cases of pituitary hyperplasia with elevated IGF-I, ectopic GHRH syndrome must be considered even if physical features of acromegaly are absent. It is also important to measure plasma GHRH concentrations in order to give a diagnosis.

Sphingosine 1-phosphate contracts canine basilar arteries in vitro and in vivo: possible role in pathogenesis of cerebral vasospasm.

BACKGROUND AND PURPOSE: Sphingosine 1-phosphate (S1P) is a platelet-derived bioactive lipid that exerts a variety of biological responses, including vasocontraction. To understand the involvement of S1P in cerebral vasospasm, we investigated the effect of S1P on vasocontraction of the canine basilar artery in vitro and in vivo. METHODS: We recorded isometric tension in basilar arterial rings from dogs in vitro and estimated time-course changes in the diameter of canine basilar arteries and the S1P concentration in cerebrospinal fluid (CSF) by angiography and radioreceptor assays, respectively, after administering S1P into the cisterna magna. Changes in the supernatant S1P concentration during clot formation were monitored by using the in vitro subarachnoid hemorrhage model, in which blood is mixed with CSF. RESULTS: At concentrations ranging between 100 nmol/L and 10 micromol/L, S1P induced a dose-dependent contraction of the basilar artery in vitro. This effect was significantly inhibited by Y-27632, a highly selective Rho-kinase inhibitor. The administration of S1P into the CSF induced a 60% to 70% decrease in the arterial diameter within 15 minutes, and vasocontraction continued for 2 days thereafter. The concentration of S1P in the supernatant during clot formation in vitro reached approximately 300 nmol/L. CONCLUSIONS: S1P induces vasocontraction in the canine basilar artery in vitro and in vivo, possibly through a mechanism involving activation of the Rho/Rho-kinase pathway. Thus, S1P might be considered as a novel spasmogenic substance involved in cerebral vasospasm after subarachnoid hemorrhage.

Sensitized mast cells migrate toward the antigen: a response regulated by p38 mitogen-activated protein kinase and Rho-associated coiled-coil-forming protein kinase.

Although mast cells accumulate within the mucosal epithelial layer of patients with allergic rhinitis and bronchial asthma, the responsible chemotactic factors are undefined. We investigated whether mast cells sensitized with Ag-specific IgE migrate toward the Ag. MC/9 mast cells sensitized with anti-DNP IgE migrated toward DNP-conjugated human serum albumin. This migration was directional, and the degree was stronger than that induced by stem cell factor. IL-3 and stem cell factor-dependent cultured mast cells derived from mouse bone marrow also migrated toward the Ag. Subsequent migration mediated by the Fc(epsilon)RI was significantly inhibited by incubating the cells with Y-27632, a Rho-associated coiled-coil-forming protein kinase inhibitor, or with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Both p38 MAPK and MAPK-activated protein kinase (MAPKAPK)2 were activated following Fc(epsilon)RI aggregation, and activation of MAPKAPK2 was almost completely inhibited by 10 microM SB203580. Wortmannin or a low concentration of SB203580 partially inhibited MAPKAPK2, but did not block mast cell migration. In contrast, Y-27632 did not affect the activation of MAPKAPK2. These results indicate that Ag works not only as a stimulant for allergic mediators from IgE-sensitized mast cells, but also as a chemotactic factor for mast cells. Both p38 MAPK activation and Rho-dependent activation of Rho-associated coiled-coil-forming protein kinase may be required for Fc(epsilon)RI-mediated cell migration.

PTHrP increases pancreatic beta-cell-specific functions in well-differentiated cells.

Parathyroid hormone-related protein (PTHrP) is reportedly produced in normal islets and insulinomas. PTHrP induces differentiation in some cell-types and growth in others. We examined whether PTHrP production is greater in well-differentiated or growing beta cells and whether PTHrP induces differentiation or growth in beta cells. We used four groups of the well-differentiated mouse beta cell line MIN6 with 17, 25, 31 and 41 passages, and mouse pancreatic islets. With passage, insulin content diminished, whereas the expression of PTHrP, its activating enzyme furin and cell growth gradually increased. PTHrP increased insulin content and mRNA levels more in MIN6-17 cells than in MIN6-41 cells. In contrast, PTHrP increased DNA synthesis more extensively in MIN6-41 cells than in MIN6-17 cells. Dibutyryl cAMP reproduced PTHrP's effect on insulin content and DNA synthesis. We conclude that PTHrP increases insulin expression in well-differentiated beta cells through the cAMP pathway and stimulates growth in growing beta cells.

Sphingosine 1-phosphate may be a major component of plasma lipoproteins responsible for the cytoprotective actions in human umbilical vein endothelial cells.

Sphingosine 1-phosphate (S1P), a novel lipid mediator, is concentrated in the fraction of lipoproteins that include high density lipoprotein (HDL) and low density lipoprotein (LDL) in human plasma. Here, we show that oxidation of LDL resulted in a marked reduction in the S1P level in association with a marked accumulation of lysophosphatidylcholine (LPC). We therefore investigated the role of the lipoprotein-associated lipids especially S1P in the lipoprotein-induced cytoprotective or cytotoxic actions in human umbilical vein endothelial cells. The viability of the cells gradually decreased in the absence of serum or growth factors in the culture medium. The addition of oxidized LDL (ox-LDL) accelerated the decrease in the cell viability. LPC and 7-ketocholesterol mimicked ox-LDL actions. On the other hand, HDL and LDL almost completely reversed the serum deprivation- or ox-LDL-induced cytotoxicity. Exogenous S1P mimicked cytoprotective actions. Moreover, the S1P-rich fraction and chromatographically purified S1P from HDL exerted cytoprotective actions, but the rest of the fractions did not. The cytoprotective actions of HDL and S1P were associated with extracellular signal-regulated kinase (ERK) activation and were almost completely inhibited by pertussis toxin and PD98059, an ERK kinase inhibitor. The HDL-induced action was specifically desensitized in the S1P-pretreated cells. Taken together, these results indicate that the lipoprotein-associated S1P and the lipid receptor-mediated signal pathways may be responsible for the lipoprotein-induced cytoprotective actions. Furthermore, the decrease in the S1P content, in addition to the accumulation of cytotoxic substances such as LPC, may be important for the acquisition of the cytotoxic property to ox-LDL.

Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells.

Exogenous sphingosine 1-phosphate (S1P) increased cytosolic Ca(2+) concentration, stimulated thymidine incorporation (DNA synthesis) and inhibited cell migration in rat aortic smooth-muscle cells (AoSMCs). Although exogenous sphingosine, a substrate of sphingosine kinase or a precursor of S1P, markedly induced the intracellular accumulation of S1P, the lipid failed to mimic the S1P-induced actions. In contrast, dihydrosphingosine 1-phosphate (DHS1P), an S1P receptor agonist, duplicated these S1P actions even though DHS1P was approx. 20-50-fold less potent than S1P. The pharmacological properties of DHS1P for the S1P receptor subtypes Edg-1, Edg-3, Edg-5 and Edg-6 were compared in Chinese hamster ovary (CHO) cells that were overexpressing the respective receptor. In these S1P-receptor-overexpressing cells, DHS1P was approx. 20-30-fold less potent than S1P for the displacement of [(3)H]S1P binding and inositol phosphate response in Edg-5-expressing CHO cells, as was the case for AoSMCs. However, it was slightly (not more than 3-fold) less potent than S1P in cells expressing Edg-1, Edg-3 or Edg-6. Of the above-mentioned four types of S1P receptor, Edg-5 was abundantly expressed in AoSMCs, as demonstrated by Northern blotting. These results suggest that the intracellular accumulation of S1P is not necessary for the S1P-induced Ca(2+) response, for the stimulation of DNA synthesis or for the inhibition of cell migration. Thus these S1P-induced actions might be mediated through extracellular (or cell-surface) S1P receptors in AoSMCs: Edg-5 might be a most important receptor subtype.

[Establishment of the method for the measurement of sphingosine-1-phosphate in biological samples and its application for S1P research].

Sphingosine 1-phosphate (S1P), one of the sphingolipid metabolites, has been shown to participate in a variety of cellular responses including proliferation, differentiation, adhesion, motility, and apoptosis. These cellular responses elicited by S1P were first thought to be mediated through an intracellular target(s), but extracellular mechanisms through G-protein-coupled S1P receptors have also been suggested. In addition to the studies examining the functions of the lipid on the cells and tissues, the measurement of the lipid concentration is also important for understanding the physiological and pathophysiological roles of the lipid. We have recently developed a novel quantitative method for measurement of S1P, which was based on the competition of S1P in the samples with the labeled S1P on the S1P receptor Edg-1. Here, we compared our method with previously published ones in several points including specificity and simplicity. We further presented our recent results obtained by using this novel quantitative method and finally mentioned the prospects of the S1P measurement in lipid research, especially in relation to several disorders.

Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions.

The concentration of sphingosine 1-phosphate (S1P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol phosphate response to plasma or serum mediated by Edg-3, one of the S1P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S1P in these samples. The inositol phosphate response to exogenous S1P was markedly attenuated in the presence of charcoal-treated low-S1P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S1P and was associated with the trapping of exogenous S1P; the inositol phosphate response to P(2)-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S1P than lipoprotein-deficient serum. Consistent with this observation, we detected a 15-100-fold higher amount of S1P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S1P is distributed in this fraction. These results suggest that the tight binding of S1P to the components of serum or plasma, including lipoproteins, may interfere with the S1P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.

Sphingosine 1-phosphate stimulates insulin secretion in HIT-T 15 cells and mouse islets.

Sphingosine is involved in the regulation of cellular processes as a second messenger in various kinds of cells. Since the possible involvement of sphingosine has not been investigated in pancreatic beta-cells, we determined the expression of putative sphingosine 1-phosphate (S1P) receptors and the effect of sphingosine on pancreatic beta-cell function using a clonal Hamster beta-cell line, HIT-T 15 cells and isolated mouse islets. We showed the expression of putative S1P receptors, Edg-3 and AGR16/H218 in HIT-T 15 cells. Ten and 20 microM S1P significantly stimulated insulin secretion for 10 minutes in HIT-T 15 cells. Ten microM S1P significantly increased insulin secretion from isolated mouse islets. Ten microM S1P obviously increased intracellular Ca2+ concentration ([Ca2+]i). Fifty nM nifedipine did not affect the S1P stimulation of insulin secretion in HIT-T 15 cells. Two microM U73122 (phospholipase C inhibitor) completely deleted 10 microM S1P-induced stimulation of insulin secretion for 10 minutes, but U73343 (an inactive analogue of U73122) did not. S1P dose-dependently inhibited intracellular cyclic AMP levels. Pretreatment with 100 ng/ml pertussis toxin (PTX) partially, but significantly attenuated an increase of insulin secretion by 10 microM S1P. These data suggested that PTX-sensitive G-protein-dependent pathway may, at least in part, be involved in an increase of non-glucose stimulated insulin secretion by S1P through the activation of phospholipase C-Ca2+ system.

Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay.

In Chinese hamster ovary cells overexpressing Edg-1, one of the sphingosine 1-phosphate (S1P) receptor subtypes, [(3)H]S1P binding was displaced by unlabeled S1P with IC(50), a half-maximal concentration to inhibit the binding, of about 20 nM. This radioreceptor binding was used for quantitative measurement of S1P. Among the various lipids employed, only sphingosylphosphorylcholine (SPC), other than S1P, practically displaced the binding; however, the potency of SPC was about 100 to 1000 times less than that of S1P. Thus, SPC bound to the S1P receptors inefficiently. Furthermore, before the application of test samples to this assay, S1P was partially purified: the lipid was extracted first into the aqueous phase and separated from other lipids under alkaline conditions, and then reextracted into the chloroform phase under acidic conditions. With this assay, we could specifically and quantitatively measure S1P from 2 to 40 pmol per assay well in biological samples including serum samples and various tissues. This assay also allowed us to measure the change in cellular S1P content in U937 cells after treatment with exogenous sphingosine.

Sphingosine 1-phosphate stimulates proliferation and migration of human endothelial cells possibly through the lipid receptors, Edg-1 and Edg-3.

Sphingosine 1-phosphate (S1P) stimulates thymidine incorporation (DNA synthesis), cell growth and cell migration in human aortic endothelial cells (HAECs). The extent of the S1P-induced responses are comparable to those stimulated by vascular endothelial growth factor, one of the most potent stimulators of angiogenesis. These responses to S1P were mimicked by dihydrosphingosine 1-phosphate, an S1P receptor agonist, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S1P also induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAP kinase). The activation of these enzymes was inhibited again by PTX and also by suramin, a non-selective receptor antagonist. S1P-induced DNA synthesis and ERK activation were inhibited by PD98059, an ERK kinase inhibitor, but not by SB203580, a p38 MAP kinase inhibitor. In contrast, cell migration and p38 MAP kinase activation, in response to S1P, were inhibited by SB203580 but not by PD98059. In HAECs, high-affinity S1P binding activity and expression of Edg-1 and Edg-3 mRNA were detected. These results suggest that S1P might be a novel angiogenesis factor and that the lipid-induced proliferation and migration of endothelial cells are possibly mediated through cell-surface S1P receptors, Edg-1 and Edg-3, which are linked to signalling pathways.

Role of insulin and serum on thyrotropin regulation of thyroid transcription factor-1 and pax-8 genes expression in FRTL-5 thyroid cells.

Thyrotropin (TSH), via its cyclic adenosine monophosphate (cAMP) signal, decreases thyrotropin receptor (TSHR) gene expression in FRTL-5 thyroid cells, whereas it increases expression of the thyroglobulin (Tg) gene. Despite the opposite effects of TSH on TSHR and Tg expression, both genes are positively controlled by thyroid transcription factor-1 (TTF-1) and evidence has accumulated that TSH can decrease TTF-1 mRNA levels. In this report, we further characterize the action of TSH on TTF-1 in order to understand its different activities on the TSHR and Tg genes better. The effect of TSH on the TSHR requires the presence of insulin and serum and we show here that also both factors are necessary for the TSH effect to decrease TTF-1 mRNA levels. The decrease is paralleled by a downregulation of TTF-1 protein levels as well as by a decrease in TTF-1/DNA complex when the TTF-1 site of the TSHR promoter was used as probe. Again, the decrease requires insulin and serum. The TSH downregulation of TTF-1 mRNA levels is due to a decrease in its transcription rate. Using a luciferase-linked chimera construct spanning 5.18 kb of the TTF-1 5'-flanking region, we show that TSH decreases TTF-1 promoter activity and that this effect depends on insulin and serum. These data contrast with the action of TSH on Tg and Pax-8 gene expression. TSH increases Pax-8 mRNA levels and the increase is evident whether insulin and serum are present or not. Moreover, this increase is paralleled by an increase in Pax-8 protein binding to an oligonucleotide derived from the C site of the Tg promoter, which can bind both TTF-1 and Pax-8. The present data thus show that TTF-1 gene expression is interdependently regulated by TSH and serum growth factors including insulin. They also show this interdependent-regulation is not duplicated in the case of Pax-8. We suggest that these differences may contribute to the distinct ability of TSH to regulate TSHR versus Tg gene expression in FRLT-5 thyroid cells.

Edg-6 as a putative sphingosine 1-phosphate receptor coupling to Ca(2+) signaling pathway.

The endothelial differentiation gene-6 (Edg-6) was recently identified as an orphan G-protein-coupled receptor. Its predicted amino acid sequence is very close to Edg family of receptor proteins whose ligand is supposed to be lysophosphatidic acid (LPA) or lysosphingolipid such as sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Transfection of the Edg-6 into Chinese hamster ovary (CHO) cells and K562 cells resulted in the appearance of high-affinity [(3)H]S1P binding activity. Among lipids employed, S1P and, even though less potent, SPC, displaced the [(3)H]S1P binding, but LPA was inactive. In Edg-6-transfected CHO cells, an increase in cytosolic Ca(2+) concentration in response to S1P or SPC was clearly enhanced without change in the LPA-induced action as compared with the vector-transfected cells. The enhancement of the Ca(2+) response was associated with a significant accumulation of inositol phosphate, reflecting activation of phospholipase C. Similar enhancement of Ca(2+) response to S1P or SPC was also observed in Edg-6-expressing K562 cells. These lipid-induced actions in CHO cells and K562 cells expressing Edg-6 were markedly suppressed by pertussis toxin treatment. We conclude that Edg-6 is one of S1P or lysosphingolipid receptors that couple to phospholipase C-Ca(2+) system through pertussis toxin-sensitive G-proteins.

Differential roles of Edg-1 and Edg-5, sphingosine 1-phosphate receptors, in the signaling pathways in C6 glioma cells.

Several cDNA encoding G-protein-coupled receptors, i.e. Edg-1,-3,-5,-6 and -8, have recently been identified as sphingosine 1-phosphate (S1P) receptors. However, the role of the respective receptor subtype has not been well defined. In C6 glioma cells, exogenous S1P induced expression of fibroblast growth factor-2 (FGF-2), a potent neurotrophic factor, which was associated with the stimulation of extracellular signal-regulated kinase (ERK) and the expression of early growth response-1 (Egr-1). S1P also stimulated phospholipase C (PLC)/Ca(2+) system and phospholipase D (PLD). In this study, we sought to identify S1P receptors responsible for these S1P-induced actions. Of five S1P receptor subtypes, Edg-1 and Edg-5 are expressed in the glioma cells, as evidenced by Northern blotting. We therefore prepared the cells overexpressing these S1P receptor subtypes and compared the intrinsic activities to stimulate these signaling pathways and their sensitivity to pertussis toxin (PTX). The potency of S1P and dihydrosphingosine 1-phosphate (DHS1P), another S1P receptor agonist, to stimulate the Edg-1 and Edg-5 receptors was also examined. We found that the intrinsic activity that stimulated ERK/Egr-1/FGF-2 system was much higher in Edg-1 than in Edg-5. Furthermore, DHS1P was as potent as S1P in activating ERK in control C6 cells, a pattern also observed in cells overexpressing Edg-1. On the other hand, the stimulation of the PLC/Ca(2+) system and PLD induced by S1P was PTX-insensitive, and the potency of S1P in activating PLD was roughly one order higher than that of DHS1P in control C6 cells; similar responsiveness to such pharmacological tools were observed in Edg-5-overexpressing cells. Taken together, these results suggest that Edg-1 may be the main receptor mediating the stimulation of ERK/Egr-1/FGF-2 system but that Edg-5 may be responsible for the stimulation of PLC-Ca(2+) system and PLD in native C6 glioma cells.

Sphingosylphosphorylcholine induces a hypertrophic growth response through the mitogen-activated protein kinase signaling cascade in rat neonatal cardiac myocytes.

The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 micromol/L), S1P (1 to 10 micromol/L), or SPC (0.1 to 10 micromol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein-coupled receptor and a MAPK signaling cascade.

Comparison of intrinsic activities of the putative sphingosine 1-phosphate receptor subtypes to regulate several signaling pathways in their cDNA-transfected Chinese hamster ovary cells.

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [(3)H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca(2+)](i) increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca(2+) responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca(2+) system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.

An adenosine receptor agonist-induced modulation of TSH-dependent cell growth in FRTL-5 thyroid cells mediated by inhibitory G protein, Gi.

Adenosine has been shown to modulate the TSH-induced DNA synthesis in FRTL-5 thyroid cells. The mechanism of this adenosine action has been somewhat controversial because both A1 adenosine receptor-mediated and non-receptor-mediated mechanisms have been proposed. We have now reexamined our preliminary finding of the inhibitory action of a non-metabolizable adenosine derivative, N6-(L-2-phenylisopropyl)adenosine (PIA), on the TSH-induced DNA synthesis to clarify the adenosine-dependent mechanism of cell growth modulation. PIA dose-dependently inhibited the TSH-induced DNA synthesis expressed by [3H]thymidine incorporation into DNA. This adenosine derivative also prevented the TSH-induced entry of the cell cycle to the S phase at 24 h of culture and the increase in cell number at 48 h. These PIA actions on different aspects of TSH-dependent cell growth were abolished by the treatment of the cells with pertussis toxin, suggesting the involvement of Gi in the PIA action mechanism. Dibutyryl cAMP-induced DNA synthesis was not influenced by PIA. In concert with our previous finding that PIA in a similar concentration range inhibited TSH-induced cAMP production through the adenosine A1 receptor, the present results strongly support the idea that the major pathway of adenosine signaling for the inhibition of the TSH-induced cell proliferation is through the A1 adenosine receptor-Gi system.

[A study on the factors affecting friendship with HIV-infected students in high school: from the survey of AIDS related knowledge, information sources and attitudes in 1989 and 1992].

To clarify the factors affecting the friendship with human immunodeficiency virus (HIV)-infected persons, a self-reported questionnaire study was conducted in two high schools in Tokyo in 1989 and 1992. A complete set of responses was obtained from 541 (98%) and 517 (99%) students in 1989 and 1992, respectively. The following results were obtained: 1) The proportion of the students who received information on HIV/acquired immunodeficiency syndrome (AIDS) from school teachers in 1992 was higher than that in 1989 for both males and females. Similarly, for both males and females, the proportion of students with correct knowledge on HIV transmission was significantly higher in 1992. The proportion of the students who intended to have a friendship with a hypothetical HIV-infected classmate or close friend in 1992 was significantly higher. 2) The results of multiple logistic regression analysis indicated that such factors as having more correct knowledge on HIV transmission and considering the confidentiality of persons with HIV/AIDS were significantly related to having friendship with a hypothetical HIV-infected classmate for both 1989 and 1992; such factors as being a female and having an increasing risk of HIV infection were significantly related to the responses obtained for 1992.

Heat stress aggravates viral myocarditis in mice.

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.

Activation of phospholipase C-Ca2+ system by sphingosine 1-phosphate in CHO cells transfected with Edg-3, a putative lipid receptor.

Sphingosine 1-phosphate (S1P) induces phospholipase C (PLC) activation and Ca2+ mobilization in many types of cells. We examined the possible involvement of Edg-3, one of the putative S1P receptors, in the phospholipase C (PLC)-Ca2+ system. S1P increased the cytoplasmic free Ca2+ concentration without detectable inositol phosphate production in vector-transfected CHO cells. In the Edg-3-transfected cells, however, the S1P-induced Ca2+ response was clearly enhanced, which was associated with a significant production of inositol phosphate. These S1P-induced responses in the Edg-3-transfected cells were inhibited by U73122, a potent PLC inhibitor. We conclude that Edg-3 may be one of the S1P receptors participating in the activation of the PLC-Ca2+ system.