Production and secretion of adrenomedullin from glial cell tumors and its effects on cAMP production

The expression of adrenomedullin (ADM) and its mRNA was studied in human glial cell tumors and cultured glioblastoma cell lines, T98G and A172. Northern blot analysis showed that ADM mRNA was expressed in all brain tumors examined (three anaplastic astrocytomas and two glioblastomas multiforme) and in the glioblastoma cell lines. Immunoreactive (IR-) ADM was detectable in these brain tumors by radioimmunoassay (0.31-2.0 pmol/g wet weight), except for one anaplastic astrocytoma. Reverse phase high performance liquid chromatography of the tumor extracts showed a single peak eluting in the position of ADM-(1-52). IR-ADM concentrations in the cultured media of T98G cells were 205.5 +/- 8.4 fmol/10(5) cells/24 h (mean +/- SEM, n = 5). Treatment of T98G cells with interferon gamma or interleukin 1 beta increased the expression levels of ADM mRNA and the IR-ADM concentrations in the cultured media, whereas tumor necrosis factor alpha decreased them in a dose-dependent manner. Treatment with synthetic ADM-(1-52) (10(-8) or 10(-7) mol/l) increased the cAMP concentrations in the cultured media of T98G cells. These findings suggest that ADM is secreted from glial cell tumors and is related to the pathophysiology of these tumors.     

Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.     

Relationship between glomerular epithelial cell injury and proteinuria in IgA nephropathy

The cost of in-hospital percutaneous transluminal coronary angioplasty (PTCA) has risen since the introduction of the coronary stent. Increased attention is now being given to the PTCA charges in Japan and a multicenter study is necessary with regard to in-hospital charges. To clarify the differences in in-hospital charges for PTCA with and without coronary stent [Stent Group and plain old balloon angioplasty (POBA) Group, respectively], we studied the PTCA charges of 352 patients in 6 hospitals. Age, male gender and extent of coronary artery disease were not different. The ratio of acute myocardial infarction ranged from 16% to 64% and that of coronary stenting ranged from 24% to 65% (p < 0.001). In-hospital charge ranged from 1.4 +/- 0.8 to 2.2 +/- 1.0 million yen (p < 0.0001). The procedural charge accounted for 53% to 75% of the in-hospital charge (p < 0.01). The in-hospital charge ranged from 1.6 +/- 0.7 to 3.3 +/- 1.6 million yen in the Stent Group, higher than the charge of 1.1 +/- 0.8 to 1.9 +/- 0.7 million yen in the POBA Group (p < 0.0001). There was a statistical difference in the number of balloon catheters used (1.1 +/- 0.4 to 2.1 +/- 0.9, p < 0.005) but not in the mean number of stents used (1.1 +/- 0.3 to 1.4 +/- 0.7). The procedural charge of the institutes with higher stenting rate (> 45%) seemed to be lower than that of the institutes with lower stenting rate (p < 0.02). In conclusion, there are large variation between institutions in PTCA charges, and in-hospital charges increased with the use of stents on introduction of the Diagnosis Related Group used in the United States. We should charge separately for coronary stenting and POBA. Despite any initial increase in the in-hospital charge for coronary stenting compared to POBA, successful stent implantation will result in a superior saving in procedural charges.     

cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification

The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like "cuboidal" morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix gamma-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC.     

Cardiovascular effects of adrenomedullin in teleost fishes

Adrenomedullin is a fifty-two-amino acid polypeptide that was first discovered in pheochromocytoma cells, and later in the normal adrenal medullae, lungs, kidneys, and blood. In mammals, adrenomedullin has vasodepressive effects, mainly by decreasing peripheral vascular resistance. I investigated the effects of adrenomedullin in fish to see if this novel neuropeptide would have an effect in lower vertebrates, or if its actions were limited to the higher vertebrates. Bolus injections of adrenomedullin resulted in a reduction of heart rate and dorsal aortic pressure in the rainbow trout Oncorhynchus mykiss. However, adrenomedullin had no effect in the Atlantic cod, Gadus morhua. The effects of adrenomedullin in trout appear to be due to a direct action on the peripheral vasculature, as pre-treatment of celiac artery strips with tetrodoxin had no effect on the ability of adrenomedullin to relax the strip.     

The role of Tec protein-tyrosine kinase in T cell signaling

The Tec protein-tyrosine kinase family includes Btk, Itk/Tsk/Emt, Tec, Rlk/Txk, and Bmx which are involved in signals mediated by various cytokines or antigen receptors. Itk is expressed primarily in T cells and activated by TCR/CD3, CD28, and CD2. However, the defect in T cell signaling in itk-deficient mice is very modest. Thus, we looked for other Tec family kinases that could be expressed in lymphoid cells and involved in T cell signal transduction. Here, we demonstrate that Tec, expressed in T cells, is activated following TCR/CD3 or CD28 ligation and interacts with CD28 receptor in an activation-dependent manner. This interaction involves the Tec SH3 domain and the proline-rich motifs in CD28. We also show that Tec can phosphorylate p62(dok), one CD28-specific substrate, whereas Itk cannot. Overexpression of Tec but not Itk can enhance the interleukin-2 promoter activity mediated by TCR/CD3 or CD28 stimulation and introduction of a kinase-dead Tec but not Itk can suppress interleukin-2 expression, indicating that Tec is directly involved in T cell activation. Altogether, these data demonstrate that Tec kinase is an integral component of T cell signaling and that the two Tec family kinases, Tec and Itk, have distinct roles in T cell activation.     

Developmental regulation of cAMP signaling pathways in thymocyte development

Major developmental transitions in thymocyte differentiation are accompanied by sharp alterations in cAMP metabolism. We have analyzed the cAMP accumulation responses of cell populations representing successive stages of T-cell development, namely: immature TcR- thymocytes from SCID mice, proliferating cortical blasts, small cortical thymocytes, medullary thymocytes and peripheral T cells. We find that all classes of thymocytes exhibit higher cAMP synthesis in response to forskolin than peripheral T cells. In immature TcR- thymocytes, this high capacity is buffered by efficient phosphodiesterase activity, but in CD4+CD8+TcRlow thymocytes, phosphodiesterase activity becomes much less effective. Phosphodiesterase activity then rises again after positive selection. The ability of thymocytes to respond to prostaglandin E is regulated distinctly from their ability to respond to forskolin. Unlike forskolin, PGE1 induces cAMP synthesis to similar levels in all classes of thymocytes, possibly due to partial activation of phosphodiesterase in cortical thymocytes by PGE1. Finally, we report a novel effect of Ca2+/protein kinase C signaling on cAMP accumulation, which occurs selectively in the proliferating cortical blasts.     

High glucose-induced mesangial cell altered contractility: role of the polyol pathway

Glomerular mesangial cells cultured in high glucose conditions display impaired contractile responsiveness. It was postulated that glucose metabolism through the polyol pathway leads to altered mesangial cell contractility involving protein kinase C. Rat mesangial cells were growth-arrested for 24 h with 0.5% fetal bovine serum in either normal (5.6 mmol/l) or high (30 mmol/l) glucose concentrations or high glucose plus the aldose reductase inhibitor, ARI-509 (100 micromol/l). The reduction of cell planar surface area (contraction) in response to endothelin-1 (0.1 micromol/l), or to phorbol 12-myristate 13-acetate (50 pmol/l), was studied by videomicroscopy. In response to endothelin-1, mesangial cells in normal glucose contracted to 52+/-3% of initial planar area. In high glucose, the significantly (p < 0.05) smaller cell size and no contractile responsiveness to endothelin-1 were normalized with ARI-509. Membrane-associated diacylglycerol, measured by a kinase specific 32P-phosphorylation assay, in high glucose was unchanged after 3 h, but significantly increased (p < 0.05) after 24 h which was normalized with ARI-509. Protein kinase C activity, measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate was: increased by 32% at 3 h of high glucose, unchanged by ARI-509; and decreased significantly (p < 0.05) at 24 h compared to cells in normal glucose, normalized by ARI-509. Total cellular protein kinase C-alpha, -delta and -epsilon, analysed by immunoblotting, were unchanged in high glucose at 24 h. Only protein kinase C-epsilon content was reduced by ARI-509 in both normal and high glucose. Therefore, high glucose-induced loss of mesangial cell contractility, diacylglycerol accumulation and altered protein kinase C activity are mediated through activation of the polyol-pathway, although no specific relationship between elevated diacylglycerol and protein kinase C activity was observed. In high glucose, altered protein kinase C function, or another mechanism related to the polyol pathway, contribute to loss of mesangial cell contractile responsiveness.     

A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway

Continuous growth and development in plants are accomplished by meristems, groups of undifferentiated cells that persist as stem cells and initiate organs. While the structures of the apical and floral meristems in dicotyledonous plants have been well described, little is known about the underlying molecular mechanisms controlling cell proliferation and differentiation in these structures. We have shown previously that the CLAVATA1 (CLV1) gene in Arabidopsis encodes a receptor kinase-like protein that controls the size of the apical and floral meristems. Here, we show that KAPP, a gene encoding a kinase-associated protein phosphatase, is expressed in apical and young floral meristems, along with CLV1. Overexpression of KAPP mimics the clv1 mutant phenotype. Furthermore, CLV1 has kinase activity: it phosphorylates both itself and KAPP. Finally, KAPP binds and dephosphorylates CLV1. We present a model where KAPP functions as a negative regulator of the CLAVATA1 signal transduction pathway.     

Mechanisms of progressive glomerular injury in membranous nephropathy

Glomerular function and structure were serially evaluated in 15 patients with membranous nephropathy who exhibited relapsing nephrosis and chronic depression of GFR. GFR declined from 56+/-8 (mean+/-SEM) at onset to 31+/-4 ml/min per 1.73 m2 after a 2- to 5-yr period of observation (P < 0.05). An analysis of filtration dynamics suggested persistent elevation of net ultrafiltration pressure. To examine a possible role for declining intrinsic glomerular filtration capacity as the basis for the observed hypofiltration, glomeruli in the baseline and a repeat biopsy (performed after a median of 28 mo) were subjected to morphometric analysis and mathematical modeling. Analysis of the baseline biopsy revealed a reduction in filtration slit frequency and thickening of the glomerular basement membrane, lowering computed hydraulic permeability by 66% compared with normal kidney donors. In contrast, filtration surface area was increased by 37% as a result of glomerular hypertrophy. The repeat biopsy revealed persistent depression of hydraulic permeability, primarily owing to foot process broadening. An additional finding was a decrease in filtration surface area from baseline in patent glomeruli, possibly due to encroachment on the capillary lumen of an increasingly widened basement membrane. Also, a striking increase in the prevalence of global glomerulosclerosis from 7+/-2% to 23+/-4% was found between the two biopsies, suggesting a significant loss of functioning nephrons. It is concluded that hypofiltration in membranous nephropathy is the consequence of a biphasic loss of glomerular ultrafiltration capacity, initially owing to impaired hydraulic permeability that is later exacerbated by a superimposed loss of functioning glomeruli and of filtration surface area.     

Autocrine role for the endothelin-B receptor in the secretion of adrenomedullin

Adrenomedullin, originally discovered in human pheochromocytoma, is a vasodilating and natriuretic peptide of vascular endothelial and smooth muscle cell origin. Although endothelin-1 (ET-1) has been implicated as a vasoconstricting and growth-promoting peptide of endothelial origin, it may more importantly function as an autocrine factor and release vasodilatory substances such as nitric oxide by mechanisms linked to the endothelin-B (ETB) receptor subtype. The present study was designed to establish that the ETB receptor stimulates the secretion of adrenomedullin from cultured canine aortic endothelial cells. We first sought to determine the presence and production of adrenomedullin in canine aortic endothelial cells using immunohistochemistry and Northern blot analysis, which revealed that adrenomedullin immunoreactivity and adrenomedullin mRNA were present in canine aortic endothelial cells. Second, adrenomedullin was time-dependently secreted from canine aortic endothelial cells, with a secretion rate of 15.7+/-1.5 pg/10(5) cells per 24 hours. Furthermore, immunohistochemistry revealed the presence of the ETB receptor in canine aortic endothelial cells, and ETB receptor stimulation by sarafotoxin S6c increased adrenomedullin production and secretion from canine aortic endothelial cells. Such actions were blocked with the ETB receptor antagonist IRL-2500 but not with ETA receptor antagonist FR-139317. These studies are the first to report an additional autocrine role of the ETB receptor in the release of vasodilating and natriuretic peptide adrenomedullin, and they suggest another important vasoactive system regulated by the ET receptor subtype.     

Cytoprotective effects of nicorandil on hypothermic injury to immature cardiac myocytes--comparison with nitroglycerin, diltiazem and prostaglandin E1

The purpose of this study was to evaluate the functional and biochemical effects of nicorandil (NRD), nitroglycerin (NTG), diltiazem (DTZ) and prostaglandin E1 (PGE) on cardiac myocytes incubated under hypothermic conditions. Cardiac myocytes were isolated from neonatal rat ventricles and cultured for 4 days with MCDB 107 medium. Myocytes (12.5 x 10(5) myocytes/flask) were then incubated at 4 degrees C for 24 h in media containing various concentrations of NRD, NTG, DTZ or PGE. After hypothermic incubation, creatine kinase (CK) and lactate dehydrogenase (LDH) were measured. The myocytes were cultured for an additional 24 h at 37 degrees C to evaluate the recovery of myocyte beating rate. In the nicorandil groups, 10(-4) M NRD showed a significantly increased beating rate recovery compared to the control (44% vs 25% respectively, as a percentage of the baseline values; i.e., beating rate prior to hypothermic incubation). Although treatment with 10(-6) M diltiazem showed no beneficial effects (10(-6) M; 25%, control; 30%), beating was not observed at all with 10(-4) M or 10(-5) M DTZ. There were no significant changes among the NTG and PGE groups. The release of CK and LDH was significantly suppressed with 10(-4) M NRD (10(-4) M: 24.1 mIU/flask, 257.2; control: 125.4, 459.5, respectively). In contrast, 10(-4) M DTZ showed significantly increased CK and LDH levels compared to its respective control (10(-4) M: 203.3 mIU/flask, 883.4; control: 112.3, 457.4, respectively). There were no significant differences in CK and LDH levels among the NTG and PGE groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of endothelial cell products in the regulation of adrenocortical function: actions of endothelin, nitric oxide, adrenomedullin and PAMP

There is evidence to suggest that the cells of the vascular endothelium secrete factors, including endothelin-1, nitric oxide, adrenomedullin and PAMP which have a role in regulating adrenal function. There is strong evidence from many studies that each of these factors is able to affect aldosterone secretion, with much less evidence relating to glucocorticoid secretion. The recent evidence suggests that both adrenomedullin and PAMP, like endothelin-1, stimulate aldosterone secretion. The controversies surrounding this issue are discussed. What emerges most strongly from the literature, however, is the evidence that, in addition to being secreted by the vascular endothelium of the adrenal, the peptides endothelin-1 and adrenomedullin are produced within the zona glomerulosa cells themselves, suggesting an autocrine as well as a paracrine role in the regulation of zona glomerulosa function and aldosterone secretion.     

The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury

BACKGROUND: Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. METHODS: The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. RESULTS: Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. CONCLUSIONS: Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.     

Cigarette smoke augments asbestos-induced alveolar epithelial cell injury: role of free radicals

This review provides a critical evaluation of the increasing use of gene therapy in the treatment of malignancies to induce active cell death (ACD, apoptosis). This approach is consistent with the notion that cancer is an anomalous accumulation of cells largely resulting from diminished cell death. The review details the main genes potentially useful for therapy. Among these, p53 has received the majority of the investigators' attention and provided encouraging results. Even greater hope is offered by newly tried direct inducers of apoptosis, such as bax, bclXs and caspases. Another fruitful direction is the association of apoptosis-inducing gene transfer with radio- and chemotherapy, which are also inducers of ACD. There is a delicate balance between cell gain through mitosis and cell loss in neoplasia because spontaneous apoptosis is widely present in tumors. In fact, the tumor environment favors bystander cell killing which appears to be a fundamental mechanism insofar as the rate of observed cell mortality cannot be accounted for by the known methods of gene transduction with efficiencies far below 100%. We conclude that apoptosis offers a mainstream approach for cancer gene therapy since ACD is highly inducible and only limited gains in malignant cell apoptosis may displace tumors from growth to regression.     

Autocrine/paracrine role of adrenomedullin in cultured endothelial and mesangial cells

Neuropsychological profiles were assessed in a large group of nondemented control subjects (n = 261) and individuals with dementia of the Alzheimer type (DAT) (n = 407) by subjecting their psychometric test results to a factor analysis. Nondemented control subjects were functionally homogeneous with only one factor accounting for the results. The results of the factor analysis on the very mild DAT and mild DAT groups, however, yielded a mental control/frontal factor, a memory-verbal/temporal factor, and a visuospatial/parietal factor. Forty-one of the original set of participants came to autopsy an average of 5.1 years after psychometric testing and had neurofibrillary tangles, total senile plaques, and cored senile plaques estimated from frontal, temporal, and parietal regions. The results of correlations indicated that the relative burden of cored senile plaques was systematically related to the three psychometric factors. These results suggest a connection between the specific functions as defined by neuropsychological measures and specific neuropathology occurring in associated areas of cortex.