Role of cytosolic phospholipase A2 in cytokine-stimulated prostaglandin release by human gallbladder cells

Eicosanoids are involved in gallbladder inflammation, epithelial water transport, and mucous secretion. Phospholipase A, enzymes liberate arachidonic acid from membrane phosphohpids for the synthesis of eicosanoids. The purpose of this study was to determine the effect of selective cytoplasmic and secretory phospholipase A; inhibitors on basal and stimulated arachidonic acid and prostaglandin F., release in gallbladder cells. Western immunoblotting was employed to evaluate both cytosolic and secretory phospholipase A₂ enzymes in human gallbladder cells. Cells were incubated tor 22 hours with H-labelcd arachidonic acid. Arachidonic acid and prostaglandin E₂ release was then measured in the supernate after 2 hours of exposure to human interleukin-lβ, alone or after pretreatment for I hour with the inhibitors. Unstimulated gallbladder cells express both H5 kDa cytosolic and 14 kDa secretory phospholipase A;. The 85 kDa phospholipase A₂ was induced by interleukin-lβ, whereas there was no apparent change in secretory phospholipase A. enzyme concentrations. Both the secretory phospholipase A. inhibitor p-bromophenylacyl bromide and the cytosolic phospholipase A, inhibitor arachidonyl trirluorometlvyl ketone decreased basal and interleukin-1 β-stunulated arachidonic acid release. In contrast, only inhibition of cytosolic phospholipase A: led to a decrease in interleukin-1 β-stimulated prostaglandin E₂: release. Basal and intcrlcukin-lβ—stimulated arachidonic acid release appears to be the result of the activity ol both cytosolic and secretory phospholipase A₂. Interleukin-Iβ—stimulated prostaglandin E₂ release appears to he dependent on the activity of cytosolic phospholipase A₂ Supported by grant DK 27695 from the United States Public Health Service. Presented in part at the American Gastroenterologie Association Research Forum. Orlando, Fla., May 18. 1999.     

Tissular prostanoid release, phospholipase A2 activity, and lipid peroxidation in pancreas transplantation

Free radical species have been implicated as important agents in ischemia-reperfusion injury associated to transplantation procedures. This study was carried out to investigate the possible relationship between phospholipase A2 activity (PLA2), lipoperoxidation, and the changes in arachidonic acid metabolism during ischemia reperfusion injury in pancreas transplantation, as well as the effect of a free radical scavenger such as superoxide dismutase on these changes. For this purpose male Lewis rat groups (n = 7) were classified as follows: group I--control; group II--syngenic pancreas transplantation after 15 min preservation in Collins solution at 4 degrees C; group III--syngenic pancreas transplantation after 18 hr preservation in the same conditions; group IV--same as III but with administration of SOD (i.v.) immediately before revascularization in the recipient rat. The results indicate that significant increases in PLA2 activity and lipoperoxide levels occur concomitantly with an increase of thromboxane B2 (TXB2) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) in pancreatic tissue after pancreas transplantation. The counteracting effect of a free radical scavenger such as SOD supports the role of oxygen free radicals (OFR) mediating activation of PLA2 and subsequent formation of eicosanoids in pancreas transplantation.     

前列腺素E2受体在前列腺素E2诱导H9c2心肌细胞肥大中的作用

目的 评价前列腺素E2受体(EP受体)在前列腺素E2(PCE2)诱导H9c2心肌细胞肥大中的作用.方法 培养H9c2心肌细胞,以4×104个/ml的密度接种于培养瓶(每瓶3ml)、24孔(每孔1 ml)或6孔(每孔2 ml)培养板.采用随机数字表法,将细胞随机分为4组(n=24):空白对照组(C组)不予任何处理,继续培养48 h;PGE2组在细胞培养液中加入PGE2(终浓度1μmol/L);AH6809组(A组)在细胞培养液中加入PGE2(终浓度1μmol/L)和AH6809(EP1及EP2受体拮抗剂,终浓度10μmol/L);GW627368X组(G组)在细胞培养液中加入PGE2(终浓度1μmol/L)和GW627368X(EP4受体拮抗剂,终浓度10 μmol/L).孵育48 h后采用免疫荧光观察心肌细胞形态,Image J医学图像分析系统测量心肌细胞直径,BCA法检测心肌细胞总蛋白含量,RT-PCR法测定胞浆ANP mRNA及BNP mRNA的表达水平.结果 与C组比较,PGE2组、A组和G组心肌细胞总蛋白含量和心肌细胞直径增加,胞浆ANPmRNA及BNPmRNA表达上调(P＜0.05).与PGE2组比较,G组心肌细胞总蛋白含量和心肌细胞直径降低,胞浆ANP mRNA及BNP mRNA表达下调(P＜0.05),A组上述各指标差异无统计学意义(P＞0.05).结论 EP4受体介导了PGE2诱导的心肌细胞肥大效应,而该效应与EP1和EP2受体无关.     

Macrophage activation decreases macrophage prostaglandin E2 release in experimental trauma

Prostaglandin E2 (PGE2) derived from macrophages following trauma may contribute to trauma-induced immunosuppression. This study evaluated the effect of glucan, a macrophage-activating agent, on macrophage PGE2 release in a murine trauma model. ICR/HSD mice were administered D5W, glucan pre-trauma, or glucan post-trauma, and subjected to hindlimb crush and amputation injury. Splenic macrophages were isolated 24 hours following trauma, cultured (24 hrs), and macrophage PGE2 levels were determined. In-vitro marrow proliferation was assessed as a measure of immune function. Crush-amputation injury increased (184%) macrophage PGE2 release. In contrast, glucan administration (pre or post) reduced PGE2 levels in macrophage supernatants (71% and 85%, respectively). A 52% decrease in in-vitro bone marrow proliferation was observed following trauma. Glucan pre- or post-trauma eliminated the suppression of bone marrow proliferation. In conclusion, macrophage-activating immunomodulators may exert beneficial effects following trauma by: 1) reducing macrophage PGE2 synthesis and release; and 2) reducing traumatic suppression of bone marrow proliferation.     

Cyclooxygenase-2, prostaglandin synthases,and prostaglandin H2 metabolism in traumatic brain injury in the rat

Inflammatory mediators are important in traumatic brain injury (TBI). The objective of the present study was to investigate the expression of cyclooxygenase-2 (COX-2), prostaglandin E (PGE) and PGD synthases, and PGH2 metabolism in two rat models of TBI. Fluid percussion injury (FPI) resulted in bilateral induction of COX-2 mRNA in the dentate gyri and the cortex, whereas controlled cortical contusion injury (CCC) induced COX-2 mRNA in the ipsilateral dentate gyrus and intensely in the cortex as judged by in situ hybridization. The induction subsided within 24 h. COX-2 immunoreactivity was detectable in these areas and persisted in the ipsilateral cortex for at least 72 h after CCC. Regions with COX-2 induction co-localized with TUNEL staining, suggesting a link between COX-2 expression and cell damage. COX-2 forms PGH2, which can be isomerized to PGD2, PGE2, and PGF2alpha by enzymatic and non-enzymatic mechanisms. In situ hybridization showed that mRNA of PGD synthase and microsomal PGE synthase were present in the choroid plexus. The microsomal PGE synthase was induced bilaterally after FPI and unilaterally after CCC. Liquid chromatography-mass spectrometry showed that low speed supernatant of normal and traumatized cortex and hippocampus transformed PGH2 to PGD2 as main product. PGD2 was dehydrated in brain homogenates to biological active compounds, for example, 15-deoxy-delta12,14-PGJ2. Thus COX-2 increases in certain neurons following TBI without neuronal induction of PGD and microsomal PGE synthases, suggesting that PGH2 may decompose to PGD2 and its dehydration products by nonenzymatic mechanisms or to PGD2 by low constitutive levels of PGD synthase.     

Parathyroid hormone-related protein stimulates prostaglandin E2 release from human osteoblast-like cells: modulating effect of peptide length.

Parathyroid hormone-related protein (PTHrP) is a potent bone-resorbing protein that frequently mediates the humoral hypercalcemia of malignancy syndrome. Since prostaglandins may mediate the bone-resorptive action of certain hormones, we examined the effect of PTHrP on prostaglandin E2 (PGE2) secretion by human osteoblast-like cells. There was low-level basal secretion of PGE2 by Saos-2 cells (8.1 +/- 0.6 pg/ml). Using four different preparations of PTHrP, it was observed that with increasing peptide length, from 36 to 141 amino acids, a significant increase in efficacy for PGE2 release was seen in these cells. All forms of PTHrP were agonists for PGE2 release, with effects seen at concentrations as low as 10(-12) M in 48 h conditioned media. The amino terminus of the molecule appeared critical for this effect since the truncated derivative PTHrP-(7-34) did not induce significant PGE2 secretion. However, the influence of peptide length could not be explained by differential activation of adenylate cyclase since [Tyr36]PTHrP-(1-36)amide was equipotent to the longest peptide preparation, PTHrP-(1-141), in stimulating cyclic AMP accumulation in the Saos-2 cells. In contrast, PTHrP-(1-141) was significantly more effective than [Tyr35]PTHrP-(1-36)-amide in inducing a rise in cytosolic calcium. Further, this effect was noted at concentrations lower than those that caused significant cyclic AMP accumulation in the Saos-2 cells. PTHrP-(1-141) induced the release of PGE2 from primary human bone cell cultures to levels entirely comparable to those seen in the Saos-2 cells. PTHrP-(1-141) also induced PGE2 release by cultured fetal rat long bones at 72 h. We conclude that the carboxy-terminal region of PTHrP has important effects on cellular signal transduction pathways and on the release of a potent bone-active cytokine, PGE2.     

Effects of mechanical compression on hypertrophic scars: prostaglandin E2 release

An alteration in the normal process of tissue repair can lead to the formation of hypertrophic scars. The best prevention and control of hypertrophy, especially in burn scars, is achieved using elastocompression, through a mechanism not yet clarified. Various studies have shown that mechanical compression induces the release of prostaglandin E2 (PGE2) and that PGE2 increases the expression of collagenases. In this study, we evaluated the effect of mechanical compression in vitro on PGE2 release in human normotrophic (NS) and hypertrophic (HS) burn scars. PGE2 basal levels in HS were significantly lower than those present in NS, and compression induced a significant increase in the release of PGE2 in HS scar in remission (7.2 times basal level) and active stages (5.88 times basal level). This increase seemed to be only partially IL-1beta-dependent. These data suggest a role for PGE2 in the process of hypertrophy remission induced by pressure therapy.     

Acetaminophen inhibits spinal prostaglandin E2 release after peripheral noxious stimulation.

BACKGROUND: Prostaglandin play a pivotal role in spinal nociceptive processing. At therapeutic concentrations, acetaminophen is not a cyclooxygenase inhibitor. inhibitor. Thus, it is antinociceptive without having antiinflammatory or gastrointestinal toxic effects. This study evaluated the role of spinal prostaglandin E2 (PGE2) in antinociception produced by intraperitoneally administered acetaminophen. METHODS: The PGE2 concentrations in the dorsal horn of the spinal cord were measured after formalin was injected into the hind paw of rats. The effect of antinociceptive doses of acetaminophen (100, 200, and 300 mg/kg given intraperitoneally) on PGE2 levels and flinching behavior was monitored Spinal PGE2 and acetaminophen concentrations were obtained by microdialysis using a probe that was implanted transversely through the dorsal horn of the spinal cord at L4. Furthermore, the effects of acetaminophen on urinary prostaglandin excretion were determined. RESULTS: Intraperitoneal administration of acetaminophen resulted in a significant decrease in spinal PGE2 release that was associated with a significant reduction in the flinching behavior in the formalin test Acetaminophen was distributed rapidly into the spinal cord with maximum dialysate concentrations 4560 min after intraperitoneal administration. Urinary excretion of prostanoids (PGE2, PGF2alpha, and 6-keto-PGF1alpha) was not significantly altered after acetaminophen administration. CONCLUSIONS: The data confirm the importance of PGE2 in spinal nociceptive processing. The results suggest that antinociception after acetaminophen administration is mediated, at least in part, by inhibition of spinal PGE2 release. The mechanism, however, remains unknown. The finding that urinary excretion of prostaglandins was not affected might explain why acetaminophen is antinociceptive but does not compromise renal safety.     

Genetic Analysis of Mesangial Matrix Expansion in Aging Mice and Identification of Far2 as a Candidate Gene

Aging of the kidney is associated with renal damage, in particular mesangial matrix expansion (MME). Identifying the genes involved in this process will help to unravel the mechanisms of aging and aid in the design of novel therapeutic modalities aimed at prevention and regression. In this study, structural changes in glomeruli of 24 inbred mouse strains were characterized in male mice at 6, 12, and 20 months of age. Haplotype association mapping was used to determine genetic loci associated with the presence of MME at 20 months. This analysis identified a significant association with a 200-kb haplotype block on chromosome 6 containing Far2. Sequencing revealed that mouse strains with MME contain a 9-bp sequence in the 5′ untranslated region of Far2 that is absent in most of the strains without MME. Real-time PCR showed a two-fold increase in the expression of Far2 in the kidneys of strains with the insert, and subsequent experiments performed in vitro with luciferase reporter vectors showed that this sequence difference causes differential expression of Far2. Overexpression of Far2 in a mouse mesangial cell line induced upregulation of platelet activating factor and the fibrotic marker TGF-β. This upregulation of MME-promoting factors may result, in part, from the FAR2-catalyzed reduction of fatty acyl-coenzyme A to fatty alcohols, which are possible precursors of platelet activating factor. Overall, these data suggest the identification of a novel pathway involved in renal aging that may yield therapeutic targets for reducing MME.Renal aging is associated with a decline in renal structure and function, making the elderly more vulnerable for superimposed stress, such as hypertension, diabetes, or AKI.^1,2 Eventually, renal aging may lead to CKD, and, ultimately, treatment with dialysis or transplantation might be needed. CKD is a major health problem, especially for the growing geriatric population.³The aging kidney shows functional changes, such as decreased GFR; reduced sodium homeostasis; and morphologic changes in glomeruli, tubuli, and interstitium.⁴ A characteristic feature of glomerular aging is the mesangial accumulation of extracellular matrix (ECM) proteins, which usually precedes glomerulosclerosis.^3,5 The mesangial cell is cardinal for glomerular function through its close interaction with both endothelial cells and podocytes.⁶ Mesangial matrix expansion (MME) might be caused by nephron loss and subsequent hyperfiltration in the functional nephrons. This may result in local glomerular hypertension and compensatory hypertrophy, which are thought to lead to cytokine and growth factor-mediated MME and, eventually, glomerulosclerosis.³The normal mesangium contains several ECM proteins, including collagen type IV, V, and VI; fibronectin; and proteoglycans.^6,7 MME is believed to result from an imbalance between synthesis of ECM components and decreased ECM degradation by matrix metalloproteinases that are under the control of specific inhibitors.⁸ Several growth-promoting factors are involved in this process, but an important promoter of ECM accumulation is TGF-β.^9,10 An age-related increase in TGF-β has been shown in the rat kidney, along with an increase in age-related structural changes such as glomerulosclerosis, MME, and interstitial fibrosis.¹¹ Different pathways seem to play a role in age-related kidney damage, and although sex and genetic background seem to be of high importance, specific genes that contribute to age-related damage of the kidney still remain to be identified.⁴Mice are an ideal species for studying the genetics of aging because they have a relatively short lifespan and share 99% of their genes with humans.^12,13 With the availability of large numbers of mouse inbred strains, haplotype association mapping (HAM) can be readily performed to identify associations between the phenotype and the haplotypes of mouse inbred strains.¹⁴ Recently, several genes involved in the age-related susceptibility for albuminuria have been identified in various strains of mice based on the albumin-to-creatinine ratio.¹⁵ This ratio, however, has limitations because it is a quantitative phenotype with an unequal distribution among individuals and is far downstream of the disease cascade. In this study we characterized MME in the kidneys of 24 inbred strains in male mice at 20 months of age, using HAM to identify genes associated with MME in these aged mice.     

Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers

BACKGROUND: The renin-angiotensin system plays a critical role in cardiovascular function, but little is known about the effects of specific cyclooxygenase 2 (COX-2) inhibition on this system in healthy humans under physiologic conditions. METHODS: Twenty-one healthy female volunteers received, in a randomized, double-blind, crossover study, celecoxib 200 mg twice a day, indomethacin 50 mg three times a day, or placebo for 4 days and a single dose, each, on day 5. On day 5 of each treatment, the following parameters were assessed with subjects in an upright position before and after administration of 20 mg furosemide intravenously: plasma renin activity (PRA), plasma aldosterone, serum and urine electrolytes, and creatinine. Index metabolites of prostanoids were analyzed by gas chromatography-tandem mass spectrometry in 24-hour urine on day 4 and in 2-hour urines before and after furosemide administration. RESULTS: Baseline and furosemide-stimulated PRA were reduced to a similar degree by celecoxib and indomethacin. Plasma aldosterone and urinary excretion of potassium showed changes consistent with the alteration of PRA. Urinary excretion rates of prostaglandin E(2), (PGE(2)), 7alpha-hydroxy-5, 11-diketotetranor-prosta-1,16-dioic acid (PGE-M), and 2,3-dinor-thromboxane B(2) (TxB(2)) were not reduced by celecoxib, whereas indomethacin led to a decrease of 40%, 45%, and 80%, respectively. Both active treatments inhibited urinary excretion of 2,3-dinor-6-keto-PGF(1alpha) and 6-keto-PGF(1alpha) by 60% and 40%, respectively. CONCLUSION: Renin-release in healthy humans with normal salt intake is COX-2 dependent. While COX-1 is critical for renal and systemic PGE(2) production, renal prostacyclin synthesis is apparently COX-2 dependent. Finally, the previously demonstrated shift of the thromboxane-prostacyclin balance toward prothrombotic thromboxane by specific COX-2 inhibition is confirmed.     

Preoperative thromboxane A2/prostaglandin H2 receptor activity predicts early graft thrombosis

Purpose: This study was carried out to determine whether early failure of infrainguinal bypass grafts is associated with increased expression of platelet thromboxane A₂/prostaglandin H₂ (TXA₂/PGH₂) receptors. A prospective correlation of preoperative platelet TXA₂/PGH₂ receptor-mediated activity with lower extremity graft patency was sought. Methods: Twenty-five patients who underwent infrainguinal bypass surgery for limb salvage were studied at an inpatient academic tertiary referral center and Department of Veterans Affairs Medical Center. Outcome measures were primary graft patency rate at 3 months, platelet TXA₂/PGH₂ receptor activity by equilibrium binding with ₁₂₅I-BOP, and aggregation to the TXA₂-mimetic U46619. Results: Preoperative platelet TXA₂/PGH₂ receptor density was higher (B_max, 3100 ± 1300 vs 1500 ± 1100 sites/platelet [mean ± SD]; p = 0.004) in the five patients who had graft thrombosis within 3 months. The EC₅₀ for U46619 was lower (26 ± 6 nmol/L vs 57 ± 30 nmol/L; p < 0.05) in these patients as well, confirming the functional effect of the increased receptor density. Early graft thrombosis was more likely in patients with a platelet TXA₂/PGH₂ receptor density greater than 3000 sites/platelet (odds ratio, 76; 95% confidence interval, 3.9 to 1500) or an EC₅₀ for U46619 less than 30 nmol/L (odds ratio, 16; 95% confidence interval, 1.4 to 180). Conclusions: Elevated platelet TXA₂/PGH₂ receptor levels and enhanced sensitivity of platelet aggregation to TXA₂ predict early arterial graft thrombosis. Specific TXA₂/PGH₂ receptor antagonism may prevent one of the mechanisms that contributes to early graft occlusion. (J Vasc Surg 1998;27:317-28.)     

Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.

BACKGROUND: There are conflicting results regarding the role of periendothelial mural cells in angiogenesis. In the current study, we investigated the role of mesangial cells (MCs) in endothelial vascularization by using a three-dimensional co-culture system in basement-membrane reconstruct gel (Matrigel). METHODS: Human umbilical vein endothelial cells (ECs) and human MCs were used. In the contact co-culture system, ECs and MCs were mixed and then plated together onto Matrigel. In the non-contact co-culture system, MCs were cultured within an intercup chamber, which prevented direct physical contact with the ECs on the Matrigel but allowed both cell types to share culture medium. To visualize ECs and MCs, the cells were labelled with two different fluorescent dyes prior to the co-culture. A capillary-like network formation was observed under a fluorescent microscope and confocal microscope, and the length of the network formation was quantified by the image analyzer. RESULTS: ECs barely formed capillary-like networks when cultured alone in growth factor-free medium. However, ECs cultured with MCs in a contact condition remarkably formed capillary-like networks (9.10+/-0.96 vs 0.20+/-0.07 mm/mm2 at 6 h, contact vs ECs alone, P<0.001). Direct contact between ECs and MCs was clearly demonstrated by confocal microscopy. Differentiation into branching capillary-like structures was also observed in the non-contact co-culture system (3.02+/-1.21 mm/mm2 at 6 h, P<0.001 vs ECs alone), but less prominently than in the contact co-culture condition. Vascular endothelial growth factor (VEGF) was secreted from MCs, as determined by enzyme-linked immunosorbent assay and immunofluorescent study. Adding neutralizing antibodies against VEGF into the co-culture system partially inhibited capillary network formation. CONCLUSIONS: Our data indicate that MCs help ECs differentiate toward vascularization, in which the direct cell-cell contact between ECs and MCs plays an important role. VEGF is a mediator in this process.     

Growth-inhibitory effect of prostaglandin D2 on mouse glioma cells

The effects of prostaglandin D2 (PGD2) on the growth of mouse malignant glioma cells were studied in vitro and in vivo. The in vitro studies consisted of various concentrations of prostaglandins (PG's) being added to cultures of mouse glioma cells. At concentrations above 2.5 micrograms/ml, PGD2 strongly inhibited the proliferation of glioma cells, whereas PGE2 had no effect at the same value. Exposure to 5.0 micrograms/ml PGD2 for more than 2 hours resulted in inhibition of glioma cell proliferation. This growth-inhibitory effect of PGD2 was related to the inhibition of DNA synthesis of the cells. The in vivo studies were performed with a subcutaneously transplanted mouse glioma model. Injection of 0.5 mg/kg PGD2 into the tumor was more effective than the same concentration given by intraperitoneal injection. In mice with intracranially transplanted glioma, daily intraperitoneal injection of 0.5 mg/kg PGD2 had no significant effect on survival time.     

Induction of macrophagic prostaglandin E2 synthesis by glioma cells

OBJECT: It has been reported that glioma cells produce prostaglandin (PG)E2, which promotes the growth of tumor cells and possesses immunosuppressive activity, and that cyclooxygenase (COX) inhibitors impede tumor growth and infiltration. Macrophages in tumor-bearing hosts are activated to produce PGE2, which induces an immunosuppressive state. Note, however, that the precise mechanism by which PGE2 induces an immunosuppressive state is still unclear. In this study, the authors investigated the mechanism of PGE2 production in glioma-bearing hosts. METHODS: The human and murine glioma cells that were studied did not produce a significant amount of PGE2. However, the coculture of human peripheral blood mononuclear cells or murine peritoneal macrophages with glioma cells or conditioned glioma medium led to the production of a large amount of PGE2. In contrast, production of tumor necrosis factor and interleukin (IL)-12p70 by macrophages and cytotoxic T lymphocyte induction were suppressed by culturing with conditioned glioma medium; this suppression was abrogated by the addition of the COX inhibitor indomethacin. The macrophagic expression of COX-2, and particularly the expression of microsomal PGE synthase (mPGES)-1, a terminal enzyme of the arachidonate cascade, was enhanced by the glioma-derived soluble factors. Furthermore, IL-12p70 production was not clearly suppressed in macrophages from mPGES-1-deficient mice. The glioma-derived soluble factors were sensitive to treatment with heat and papain. CONCLUSIONS: These results indicated that PGE2 production by macrophages is enhanced by glioma-derived soluble factors, which induce an immunosuppressive state in glioma-bearing hosts. Therefore, the inhibition of PGE2 synthesis, targeting COX-2 and mPGES-1, is an effective treatment for the induction of antiglioma immune responses.     

Electrical stimulation-induced release of beta-endorphin from genetically modified neuro-2a cells

The quantity of therapeutic gene products released from genetically engineered cells can be controlled externally at different levels. The widely used approach of controlling expression, however, generally has the disadvantage that chemical substances must be applied for stimulation. An alternative strategy aims at controlling gene products at posttranslational levels such as secretion. The secretion of a therapeutic agent can be regulated if the agent is targeted to the regulated secretory pathway and stored in the secretory granules until its release. In this article we address the question of whether the release of beta-endorphin, an opioid with a potent analgesic effect, could be induced by electrically stimulating stably transfected Neuro-2a cells. Throughout this study we used the human proopiomelanocortin (POMC) gene, which is the precursor molecule for human beta-endorphin. We analyzed its subcellular localization and found it in the regulated secretory pathway in Neuro-2a cells. Using electrical field stimulation we were able to identify a stimulation pattern that significantly increased the release of beta-endorphin-immunoreactive material, although to a limited extent. This result indicates that electrical stimulation of secretion could be used to manipulate the amount of a therapeutic agent released from transplanted cells.     

Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients

Diabetic nephropathy leading to kidney failure is a major complication of both type I (insulin-dependent) and type II (non-insulin-dependent) diabetes mellitus, and glomerular structural lesions (especially expansion of the mesangium) may constitute the principal cause of decline in kidney function experienced by a significant fraction of diabetic patients. Although the biochemical bases of these mesangial abnormalities remain unknown, an understanding of the natural history of diabetic nephropathy from a combined structural and functional approach can lead to greater pathophysiological insight. Work in animals has supported the concept that the metabolic disturbances of diabetes mellitus cause diabetic nephropathy, with structural and functional lesions prevented or reversed with improved or normalized glycemic control. Additional research must address this fundamental issue in humans, especially the response of advancing mesangial lesions to improved glycemic control. Factors not directly related to the metabolic perturbations of diabetes may serve to accelerate or diminish the pathophysiological processes of diabetic nephropathy. The elucidation and management of these factors, when coupled with improved glycemic control, may moderate the development or progression of diabetic kidney lesions in humans.     

Effects of prostaglandin E2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout mice

Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-kappaB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2-/-) and one line of EP4 knockout mice (EP4-/-) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2-/- cells and markedly reduced in EP4-/- cells. PGE2 increased RANKL mRNA in EP2-/- cells and EP4-/- cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2-/- and EP4 -/- cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2-/- or EP4-/- cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2.