Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17ß exposure in ovariectomized ewes.

Estradiol-17ß (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

Urokinase plasminogen activator receptor induced non-small cell lung cancer invasion and metastasis requires NHE1 transporter expression and transport activity.

BACKGROUND: Non-small cell lung cancers (NSLC) are aggressive cancers that are insensitive to chemotherapies and accounts for nearly 33% of all cancer deaths in the United States. Two hallmarks of cancer that allow cells to invade and metastasize are sustained proliferation and enhanced motility. In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na(+)/H(+) exchanger isoform 1 (NHE1) expression and activity. METHODS AND RESULTS: The addition of 10nM uPA increased the carcinogenic potential of three NSCLC cell lines, NCI-H358, NCI-H460, and NCI-H1299. This included an increase in the rate of cell proliferation 1.6 to 1.9 fold; an increase in the percentage of cells displaying stress fibers 3.05 to 3.17 fold; and an increase in anchorage-independent growth from 1.64 to 2.0 fold. In each of these cases the increase was blocked when the experiments were performed with NHE1 inhibited by 10 µM EIPA (ethylisopropyl amiloride). To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full-length uPA compared to the uPA amino terminal fragment (ATF). Comparing uPA and ATF signaling in H460 cells, we found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased matrix metalloproteinase 9 (MMP9) activity 5.44 fold compared to 2.81 fold for ATF. To expand this signaling study, two new cell lines were generated, one with reduced NHE1 expression (H460 NHE1 K/D) and one with reduced uPAR expression (H460 uPAR K/D). Using the K/D cell lines we found that neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies in athymic mice we found that when mice were injected with H460 cells 80% of mice formed tumors with an average volume of 390 mm(3). This was compared to 20% of H460 uPAR K/D injected mice forming tumors with an average volume of 15 mm(3) and 10% of H460 NHE1 K/D injected mice forming tumors with an average volume of 5 mm(3). CONCLUSION: Taken together, these data demonstrate that uPA/uPAR-mediated tumor progression and metastasis requires NHE1 in NSCLC cells and suggests a potential therapeutic approach to blocking cancer progression.

A transcriptionally permissive epigenetic landscape at the vasoactive intestinal peptide receptor-1 promoter suggests a euchromatin nuclear position in murine CD4 T cells.

T cells express receptors for neuropeptides that mediate immunological activities. Vasoactive intestinal peptide receptor-1 (VPAC1), the prototypical group II G protein coupled receptor, binds two neuropeptides with high-affinity, called vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide. During T cell signaling, VPAC1 mRNA expression levels are significantly downregulated through a Src kinase dependent mechanism, thus altering the sensitivity for these neuropeptides during an immune reaction. Presently, it is unknown whether the mechanism that regulates VPAC1 during T cell signaling involves epigenetic changes. Therefore, we hypothesized that the epigenetic landscape consisting of diacetylation at H3K9/14 and trimethylation at H3K4, two transcriptionally permissive histone modifications, would parallel VPAC1 expression showing high enrichment in untreated T cells, but lower enrichment in alpha-CD3 treated T cells. To this end, quantitative chromatin immunoprecipitation (ChIP) analysis of H3K9/14ac and H3K4me3 was conducted using purified CD4(+) T cells, with CD45R(+) B cells as a negative control. Our data revealed that these histone modifications at the VPAC1 promoter did indeed parallel its mRNA levels between T and B lymphocytes, but did not decrease during T cell signaling. Collectively, these data strongly imply a euchromatin nuclear position for the VPAC1 locus irrespective of the activation status of T cells.

Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC(2) receptor for vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) and its G protein-coupled receptors, VPAC(1)R and VPAC(2)R, are prominent in the immune system and regulate many aspects of T cell-dependent immunity. In mouse T cells, VPAC(1)R is expressed constitutively, whereas VPAC(2)R is induced by immune stimuli. VPAC(2)R-null (VPAC(2)R(-/-)) mice on a C57BL/6 background are shown here to have normal basic immune characteristics, including serum Ig concentrations, blood levels of all leukocytes, and spleen number of total T cells (CD3(+)) and T cells bearing CD4, CD8, and CD28. Hapten-evoked cutaneous delayed-type hypersensitivity (DTH) was significantly enhanced in VPAC(2)R-null mice compared with age- and sex-matched wild-type mice. In contrast, generation of IgE anti-hapten antibodies and active cutaneous anaphylaxis were > or =70% lower in VPAC(2)R-null mice than in wild-type controls. Cytokine production by splenic CD4(+) T cells, stimulated with adherent anti-CD3 plus anti-CD28 antibodies, revealed higher levels of IL-2 (mean = 3-fold) and IFN-gamma (mean = 3-fold), and lower levels of IL-4 (mean = one-fifth) in VPAC(2)R-null mice than wild-type controls. Loss of VIP-VPAC(2)R maintenance of the normal ratio of Th2/Th1 cytokines thus leads to a state of enhanced DTH and depressed immediate-type hypersensitivity, which may alter both host defense and susceptibility to immune-mediated diseases.

Allergic diathesis in transgenic mice with constitutive T cell expression of inducible vasoactive intestinal peptide receptor.

Vasoactive intestinal peptide (VIP) and its G-protein-coupled receptors (VPAC1 and VPAC2 Rs) are prominent in the immune system. In T cells, VPAC1 R is expressed constitutively whereas VPAC2 R is induced only after stimulation of the T cell receptor (TCR) or exposure to some cytokines. VPAC1 R and VPAC2 R also transduce different effects of VIP on T cells. Constitutive expression of VPAC2 R selectively in CD4+ T cells (helper-inducer Th cells) of transgenic (TG) C57BL/6 mice directed by the lck tyrosine kinase promoter is now shown to evoke production of more Th2-type interleukins 4 and 5, and less Th1-type interferon gamma after TCR activation. VPAC2 R TG mice consequently have significant elevations of blood IgE, IgG1, and eosinophils. VPAC2 R TG mice also show increased IgE antibody responses, which mediate heightened cutaneous allergic reactions, and have depressed delayed-type hypersensitivity. VIP enhancement of the ratio of Th2 cell to Th1 cell cytokines thus evokes an allergic state in normally nonallergic mice, which suggests the possibility of neuropeptide contributions to immune phenotypic alterations in human hypersensitivity diseases.

Phospholipase A(2) activity, heat shock protein, and superoxide dismutase in rat remnant kidney.

Male Sprague-Dawley rats (150-200 g) were randomly assigned to sham operation (n=6) or 5/6 nephrectomy (n=12) procedures. Two weeks after the completion of the 5/6 nephrectomy, these animals were again randomly assigned to two groups: non-treatment or treatment with vitamin E supplementation at 200 IU/kg chow. Two weeks later, all animals were sacrificed and the kidneys harvested. The secretory phospholipase A(2) (PLA(2)) activity was elevated (150%) in the untreated remnant kidney but returned to sham values in the vitamin E-treated kidneys. The cytoprotective heat shock protein (HSP70) and the intracellular antioxidant superoxide dismutase (MnSOD, Cu/ZnSOD) were similar in sham, remnant, and vitamin E-treated remnant kidneys. We conclude that the sudden reduction of renal mass secondary to the 5/6 nephrectomy procedure stimulates PLA(2) activity but not HSP70, MnSOD, or Cu/ZnSOD. This increased activity of PLA(2) in the remnant kidney returned to sham values after vitamin E treatment. The intrinsic cellular antioxidant enzymes, MnSOD, Cu/ZnSOD, as well as the cytoprotective heat shock protein HSP70, showed no significant changes in either vitamin E-treated or untreated kidneys compared with sham. These data are suggestive that the elevation of PLA(2) is a specific and localized response to the sudden reduction of renal mass.

Vasoactive intestinal peptide mediation of development and functions of T lymphocytes.

The first phase in investigating neural regulation of immunity has delineated anatomical connections, shared mediators and receptors for mediators with distinctive effects, and the immune functional consequences of altering relevant neural activities. Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP) are represented prominently in immune organs. They have potent novel effects on many aspects of immunity, are derived from and serve as autacoids in some sets of immune cells, and they participate in both physiological and pathological immune responses. The present phase of neuroimmune research has begun to elucidate the genetic determinants of expression and functions of neuromediators in immunity. Our evolving understanding of the novel mechanisms for adaptation and specificity in the VIP/PACAP neuroimmune network suggests the importance of immunoselective transcriptional control of expression of VIP/PACAP receptors in T cells, a dominant role for numerous cytokines, and the critical involvement of small subsets of VIP-/PACAP-responsive thymocytes and T cells.

Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells.

Inflammatory cytokines, interleukin 1beta and tumor necrosis factor-alpha, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A(2) (PLA(2)). Cytokine-induced up-regulation of PLA(2) has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kappaB (NF-kappaB), suggesting a role for NF-kappaB in the regulation of group IIA PLA(2) expression. Reactive oxygen species such as H(2)O(2), which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-kappaB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-alpha has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA(2) up-regulation by attenuating NF-kappaB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA(2) mRNA and extracellular enzymatic activity over time. DPI (0.02-20 microM), added 90 min before cytokine activation, inhibited both group IIA PLA(2) mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-kappaB binding to one of two NF-kappaB consensus elements in the rat group IIA PLA(2) promoter and also was suppressed by DPI pretreatment. Antibodies to NF-kappaB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-kappaB species as the classical p50/p65 heterodimer.

Development and use of ELISA to quantify type II phospholipase A2 in normal and uremic serum.

Previously we reported that uremic plasma contained eight times more phospholipase A2 (PLA2) activity than control plasma (Costello et al., Clin Chem 1990;36:198-200). That study, however, did not distinguish between various PLA2s that could contribute to the observed increase. Therefore, we developed a sandwich ELISA to specifically quantify serum type II PLA2. By ELISA, uremic sera contained significantly more type II PLA2 than control sera (median = 1025 micrograms/L, range = 52-3320 micrograms/L vs median = 9.2 micrograms/L, range = 4.6-17.5 micrograms/L; P = 0.002). When serum samples were incubated with 1-[14C]oleate-labeled autoclaved Escherichia coli, activity was increased 14.6-fold in uremic vs normal serum, with a median of 6.5 mumol/min per liter (range 1.1-16.3) vs a control median of 0.49 mumol/min per liter (range 0.32-0.60; P = 0.002). Thus, ELISA detects about eightfold more immunoreactive type II PLA2 in uremic serum than does enzymatic analysis. Evidently, the increase in PLA2 activity previously observed in uremic plasma is primarily due to increased concentrations of type II PLA2.

Regulated expression of monocyte chemoattractant protein-1 in normal human osteoblastic cells.

An influx of monocytes is observed in many different physiological and pathophysiological states, including bone remodeling and injury. These cells appear at early stages of bone formation and repair and persist throughout the later stages. In experiments described here, unstimulated normal human osteoblastic cells did not produce detectable levels of monocyte chemotactic activity. However, interleukin-1 (IL-1)-stimulated normal human osteoblastic cells produced a chemoattractant that is similar to monocyte chemoattractant protein-1 (MCP-1) at the levels of mRNA expression, protein production, and chemotactic activity. Northern blot analysis indicates that IL-1 elicits a dose-dependent increase in MCP-1 mRNA in normal human osteoblastic cells. Two proteins of M(r) 9,000 and M(r) 13,000 were specifically immunoprecipitated with MCP-1 antiserum from IL-1-stimulated normal human osteoblastic cells. Monocyte chemotactic activity from IL-1-treated cells was blocked by MCP-1 antiserum. These studies establish that normal human osteoblastic cells can be induced to produce monocyte chemoattractants and that this is accounted for by the induced expression of MCP-1.