Regulation of vasopressin secretion by ETA and ETB receptors in compartmentalized rat hypothalamo-neurohypophysial explants

The endothelins (ET) have been implicated in vasopressin (AVP) release in vivo and in vitro. The effects of ET in this system are complex, and the net AVP secretory response likely depends on a unique combination of ET isoform, ET receptor subtype, and neural locus. The purpose of these studies was to examine the role of ET receptor subtypes at hypothalamic vs. neurohypophysial sites on somatodendritic and neurohypophysial AVP secretion. Experiments were done in cultured explants of the hypothalamo-neurohypophysial system of Long Evans rats. Either the whole explant (standard) or only the hypothalamus or posterior pituitary (compartmentalized) was exposed to log dose increases (0.01-10 nM) of the agonists ET-1 (ET(A) selective), ET-3 (nonselective), or IRL-1620 (ET(B) selective) with or without selective ET(A) (BQ-123, 2-200 nM) or ET(B) (IRL-1038, 6-600 nM) receptor antagonism. In standard explants, ET-1 and ET-3 dose-dependently increased, whereas IRL-1620 decreased net AVP release. Hypothalamic ET(B) receptor activation increased both somatodendritic and neurohypophysial AVP release. At least one intervening synapse was involved, as tetrodotoxin blocked the response. Activation of ET(A) receptors at the hypothalamic level inhibited, whereas ET(A) receptor activation at the posterior pituitary stimulated, neurohypophysial AVP secretion. Antagonism of hypothalamic ET(A) receptors potentiated the stimulatory effect of ET-1 and ET-3 on neurohypophysial secretion, an effect not observed with ET(B) receptor-induced somatodendritic release of AVP. Thus the response of whole explants reflects the net result of both stimulatory and inhibitory inputs. The integration of these excitatory and inhibitory inputs endows the vasopressinergic system with greater plasticity in its response to physiological and pathophysiological states.     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Regulation of functional differentiation of the placental villous syncytiotrophoblast by estrogen during primate pregnancy.

By using the baboon as an in vivo model for the study of the endocrinology of human pregnancy, studies in the authors' laboratories have shown that the primate placenta is an estrogen target tissue and that estrogen, via interaction with the estrogen receptor, regulates functional differentiation of the syncytiotrophoblast, which is manifest as an upregulation of key components of the progesterone biosynthetic pathway and the metabolism of corticosteroids critical to placental-fetal development. Thus, estrogen exerts specific stimulatory effects on the receptor-mediated uptake of low density lipoprotein by, and expression of, the P-450 cholesterol side-chain cleavage enzyme within the syncytiotrophoblast, thereby promoting the production of progesterone. Concomitantly, there is an estrogen-dependent developmental regulation of the 11beta-hydroxysteroid dehydrogenase enzyme system in the syncytiotrophoblast, which enhances transplacental oxidation of maternal cortisol to cortisone and leads to maturation of the fetal hypothalamic pituitary adrenocortical axis late in gestation. Consequently, estrogen has a central, integrative role in modulating the dialogue and signaling system operating between the placenta and fetus that results in the maintenance of pregnancy and the development of adrenocortical self-sufficiency that are essential for maturation of the fetus and neonatal survival after birth.     

Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets

Low oxygen (O(2)) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O(2)). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.     

The influence of oxygen and tumor necrosis factor-alpha on the cellular kinetics of term placental villous explants in culture.

Explanted placental fragments may provide a more physiological in vitro model of component cell function than single cell isolates. We have characterized these fragments for cell turnover and have monitored responses from 14 normal placentas under conditions of exogenous TNFalpha and atypical oxygen concentrations (3% and 17%), conditions associated with abnormal pregnancy and an aberrant in utero environment. Explants were assessed for apoptotic morphology, immunolocalization of Mib-1 (a proliferation marker), caspase 3 activity (an apoptosis promoter), lactate dehydrogenase (a necrosis marker), and human chorionic gonadotrophin [hCG, a marker of cytotrophoblast (CT) differentiation]. Consistent with a reduction in hCG, explants under 17% O(2) (with and without TNFalpha) showed a progressive degeneration of syncytiotrophoblast (ST) (days 0-2) followed by a restoration of hCG (days 4-8) localized to newly differentiated but not syncytialized CTs. In 3% O(2), hCG showed the same initial decline but failed to recover thereafter. Proliferation dropped significantly in 17% O(2) but was restored and exaggerated sixfold in 3% O(2). All reductions in hCG were associated with cell death and caspase-3. Early apoptosis was linked with syncytial loss; later apoptosis (days 8-11) was localized to the non-ST. Prolonged exposure to TNFalpha (days 4-11) increased ST apoptosis and necrosis but 3% O(2) had no significant effect. These findings show that placental explants can accommodate many aspects of CT proliferation, differentiation, and ST apoptosis in culture. TNFalpha enhanced ST decline but 3% oxygen (compared with 17%) was associated with reduced CT differentiation and a strong shift towards proliferation. These outcomes may reflect previous morphological changes in compromised pregnancies and confirm a possible role for oxygen and TNFalpha in aberrant trophoblast turnover.     

Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.     

Regulation of lipid metabolism by dipyridamole and adenosine antagonists in rat adipocytes

1. Acute effects of dipyridamole, an inhibitor of adenosine transport, direct activators of adenylate cyclase and thirteen adenosine antagonist analogs on fatty acid synthesis have been examined in terms of the control of [1-14C]acetate incorporation into labeled fatty acids in the presence of glucose. 2. This monosaccharide acts as a stimulator of lipogenesis by generating NADPH for the lipid synthesis. 3. The relationship between lipogenesis and lipolysis was compared with a variety of adenylate cyclase stimulators. 4. The data obtained reveals that dipyridamole potentiated the inhibitory or stimulatory effects of isoproterenol and forskolin on lipogenesis and on lipolysis, respectively. 5. In these cases the data show that it exists an inverse relationship between lipogenesis and lipolysis. 6. Dipyridamole and methylxanthine analogs only moderately affect the rate of lipolysis whereas its effects are more potent on lipogenesis and lend further support to the hypothesis that dipyridamole antagonize adenosine actions as well as methyl xanthines. 7. These results suggest that dipyridamole and adenosine antagonists alter lipogenesis independently of the lipolytic process and that it exists an inverse relationship between lipogenesis and lipolysis under some conditions whereas there are not under others.     

Regulation of adenosine receptors expression in rat B lymphocytes by insulin

Development of diabetes is associated with altered expression of adenosine receptors (ARs). Some of these alterations might be attributed to changes in insulin concentration. This study was undertaken to investigate the possible insulin effect on ARs level, and to determine the signaling pathway utilized by insulin to regulate the expression of ARs in rat B lymphocytes. Western blot analysis of B lymphocytes protein extracts indicated that all four ARs were present at detectable levels in the cells cultured for 24 h without insulin (≤10^?11 M), although the protein band of A_2A‐AR was barely visible. Inclusion of insulin (10^?8 M) in the culture medium resulted in an increase of A₁‐AR and A_2A‐AR protein levels and a significant decrease of A_2B‐AR protein, whereas the protein level of A₃‐AR remained unchanged. Alterations in the ARs protein content were accompanied by changes in the ARs mRNA levels. Increase of the insulin concentration from 10^?11 to 10^?8 M resulted in 50% decrease of A_2B‐AR mRNA level and two‐, and threefold increase of A₁‐AR and A_2A‐AR mRNA levels, respectively. Pretreatment of B cells with cycloheximide completely blocked the insulin action on A₁‐AR and A_2A‐AR mRNA, but not on A_2B‐AR expression. Detailed pharmacological analysis demonstrated that insulin‐induced A₁‐AR and A_2A‐AR mRNA expression through the Ras/Raf‐1/MEK/ERK pathway. The insulin effect on A_2B‐AR expression was blocked by p38 MAP kinase inhibitor (SB 203580). Concluding, elevated insulin concentration differentially affects the expression of ARs in B lymphocytes in a fashion that might enhance the various immunomodulatory effects of adenosine. J. Cell. Biochem. 109: 396–405, 2010. © 2009 Wiley‐Liss, Inc.     

Reduced placental docosahexaenoic acid levels associated with increased levels of sFlt-1 in preeclampsia

Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p<0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p<0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p<0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.     

Regulation of adiponectin secretion by endothelin-1

Adiponectin is an adipocyte-derived hormone best known for its insulin-sensitizing ability. The expression and circulating concentration of adiponectin are decreased in type 2 diabetics and increase following treatment with thiazolidinediones. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide whose levels are elevated in numerous disease states, including obesity and diabetes. ET-1 has profound effects on adipose tissue metabolism and alters the release of adipose-derived factors such as leptin and resistin, therefore we investigated the role of ET-1 in adiponectin secretion. 3T3-L1 adipocytes were treated with insulin (100 nM), ET-1 (100 nM), or the appropriate vehicle and adiponectin secretion into the media was determined by immunoblotting and densitometric analysis. Adiponectin secretion significantly increased 1h following insulin or ET-1 treatment, respectively. Pretreatment with ET-1 for 24h significantly inhibited the ability of insulin or ET-1 to acutely stimulate adiponectin secretion. The specific ET(A) receptor antagonist, BQ-610 (1 microM), significantly inhibited ET-1-stimulated adiponectin secretion. In summary, ET-1 acutely stimulates adiponectin secretion through the ET(A) receptor. Chronic exposure to ET-1 dramatically decreases the stimulatory effect of insulin and ET-1 on adiponectin secretion. Our findings suggest vascular factors such as ET-1 may play a role in the regulation of adiponectin secretion and whole body energy metabolism.     

Effects of glucose, glycerol, 3-hydroxybutyrate, insulin, and leptin on placental growth hormone secretion in placental explants

Placental growth hormone (PGH) is secreted from the syncytiotrophoblast in increasing amounts during pregnancy. The physiology and regulation of PGH is not well known; however, low glucose levels appear to stimulate PGH liberation IN VITRO and IN VIVO. PGH appears to have lipolytic effects, and inverse correlations between maternal body mass index and serum PGH levels have been reported. Therefore, substances related to maternal adipose tissue metabolism could influence PGH secretion. The effect of insulin, glycerol, 3-hydroxybutyrate (3-OHB), and leptin on PGH and human placental lactogen (hPL) secretion from cultured placental explants was studied. In glucose-free media, PGH content increased upto 237.5+/-28.4% of control media (p<0.001, ANOVA). Insulin levels were without effect on PGH secretion, as were 3-OHB, leptin, and glycerol at 0.02 mmol/l. Glycerol at 0.2 mmol/l increased PGH in all of the placental explants studied (n=8; mean increase 27.3+/-7.1%), and this difference was significantly different from the control explants (p=0.004). The liberation of hPL to culture media was different from PGH and was influenced by glucose and insulin. In conclusion, the absence of glucose profoundly increased PGH secretion in cultured placental explants. Addition of glycerol in physiologically relatively high concentrations showed a less pronounced stimulatory effect.     

Regulation of adenylate cyclase by adenosine and other agonists in rat myocardial sarcolemma

The presence of adenosine receptors coupled to adenylate cyclase in rat heart sarcolemma is demonstrated in these studies. Heart sarcolemma was isolated by the hypotonic shock-Lithium bromide treatment method. This preparation contained negligible amounts (2-4%) of contamination by other subcellular organelles such as mitochondria, sarcoplasmic reticulum, and myofibrils as verified by electron microscopic examination. In addition this preparation was also devoid of endothelial cells, since angiotensin-converting enzyme activity was not detected in this preparation. N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyladenosine (PIA), and adenosine N'-oxide (Ado N'-oxide) were all able to stimulate adenylate cyclase in heart sarcolemma, but not in crude homogenate, with an apparent Ka of 3-7 microM. The activation of adenylate cyclase by NECA was dependent on the concentrations of metal ions such as Mg2+ or Mn2+. The maximal stimulation was observed at lower concentrations of the metal ions (0.2-0.5 mM). At 5 mM Mg2+ or Mn2+, the stimulation by NECA was completely abolished. The stimulatory effect of NECA on adenylate cyclase was also dependent on guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In addition, 2'-deoxyadenosine showed an inhibitory effect on adenylate cyclase. The myocardial adenylate cyclase was also stimulated by beta-adrenergic agonists, dopamine and glucagon, and inhibited by cholinergic agonists such as carbachol and oxotremorine. The stimulation of adenylate cyclase by NECA was found to be additive with maximal stimulation obtained by epinephrine. These data suggest that rat heart sarcolemma contains adenosine (Ra), beta-adrenergic, dopaminergic, glucagon, and cholinergic receptors, and the stimulation of adenylate cyclase by epinephrine and adenosine occurs by distinctly different mechanism or adenosine and epinephrine stimulate different cyclase populations.     

Regulation of protein secretion by crinophagy in perfused rat liver.

We examined the secretion of three serum proteins, albumin (RSA), alpha 2 mu-globulin (alpha 2 mu G), and transferrin (Trf), in the isolated perfused liver. Within 4 h of perfusion, only 20 to 35% of previously synthesized proteins were secreted by the liver into the recirculating medium. Low temperature inhibited the secretion of alpha 2 mu G and Trf, but not RSA. The amount of RSA secreted by the liver increased twofold in the presence of leupeptin, a proteinase inhibitor, or primaquine, a weak base capable of neutralizing acidic compartments. Neither drug affected Trf secretion, while the release of alpha 2 mu G was enhanced threefold by primaquine treatment. Only 55 to 70% of the total amount of these serum proteins present in the liver at the onset of perfusion could be accounted for after 4 h of perfusion. Our evidence suggests that these losses are due to protein degradation. The degradation of RSA and alpha 2 mu G was inhibited at 15 degrees C and by both leupeptin and primaquine. Contrary, RSA degradation was not altered when livers were perfused at 20 degrees C. Morphological techniques combined with immunological probes were utilized to identify possible intracellular sites of RSA degradation. RSA and cathepsin L were colocalized to large vacuoles found near the cell periphery. Entry of RSA into these vacuoles occurred at 20 degrees C but not at 15 degrees C. Our results using perfused rat livers suggest that as much as 40% of hepatic serum proteins are degraded via fusion of secretory vesicles with lysosomes (e.g., crinophagy).     

Impaired VEGF and nitric oxide signaling after nitrofen exposure in rat fetal lung explants

We hypothesized that abnormal fetal lung growth in experimental congenital diaphragmatic hernia after maternal nitrofen exposure alters lung structure due to impaired VEGF signaling, which can be reversed with VEGF or nitric oxide (NO) treatment. Timed-pregnant Sprague-Dawley rats were treated with nitrofen on embryonic day 9 (E9), and fetal lungs were harvested for explant culture on E15. Explants were maintained in 3% O2 for 3 days and were treated with NO gas or recombinant human VEGF protein for 3 days. To determine the effects of VEGF inhibition on lung structure, normal fetal lung explants were treated with SU-5416, a VEGF receptor inhibitor, with or without exogenous NO or VEGF. We found that nitrofen treatment impaired lung structure, as evidenced by decreased branching at day 0, but lung structure was not different from controls after 3 days in culture. Nitrofen reduced lung VEGF but not endothelial NO synthase protein level. Treatment with NO enhanced lung growth in control and nitrofen-exposed lungs; however, the response to NO in the nitrofen-treated lungs was reduced when compared with controls. VEGF treatment did not cause a further increase in lung complexity after nitrofen exposure. SU-5416 treatment altered lung structure, which improved with NO but not VEGF treatment. Both nitrofen and SU-5416 treatment increased apoptosis in the mesenchyme of fetal lung explants. We conclude that nitrofen exposure increased apoptosis, decreased lung growth and reduced VEGF expression, and that exogenous NO but not VEGF treatment enhances lung growth. Disruption of lung architecture after VEGF receptor blockade was similar to nitrofen-induced changes but was more responsive to NO.