Blood pressure response to central and/or peripheral inhibition of phenylethanolamine N-methyltransferase in normotensive and hypertensive rats

We studied the effects on blood pressure and heart rate of two different phenylethanolamine N-methyltransferase (PNMT) inhibitors in normotensive, in two-kidney renal hypertensive, and in deoxycorticosterone-salt (DOC-salt) hypertensive rats. One compound (SK&F 64139) blocks the conversion of norepinephrine to epinephrine in both the central and the peripheral nervous system, whereas the other (SK&F 29661) does not cross the blood-brain barrier and therefore is active mostly in the adrenal glands. In the rats given SK&F 29661, practically no acute blood pressure changes were in the adrenal glands. In the rats given SK&F 64139 induced only a minor blood pressure and heart rate response in normotensive and two-kidney renal hypertensive rats. However, in DOC-salt hypertensive rats, it reduced arterial pressure to approximately normal levels and concomitantly slowed pulse rate. There was a close correlation between the magnitude of the blood pressure response observed in all SK&F 64139-treated animals and the control plasma norepinephrine (4 = -0.795, P less than 0.001) and epinephrine (r = -0.789, P less than 0.001) levels. These results suggest an important role for central epinephrine in regulating the peripheral sympathoadrenomedullary and the baroreceptor reflex activity, particularly when the maintenance of the high blood pressure is not renin-dependent.

Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids.

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic beta cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the alpha subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased ( approximately 55 percent) in the presence of palmitic acid, but increased ( approximately 180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic beta cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.

Renal effects of selective cyclooxygenase-2 inhibition in normotensive salt-depleted subjects.

PURPOSE: To compare the renal hemodynamic and tubular effects of celecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2) to those of naproxen, a nonselective inhibitor of cyclooxygenases in salt-depleted subjects. METHODS AND SUBJECTS: Forty subjects were randomized into four parallel groups to receive 200 mg celecoxib twice a day, 400 mg celecoxib twice a day, 500 mg naproxen twice a day, or a placebo for 7 days according to a double-blind study design. Blood pressure, renal hemodynamics, and urinary water and electrolyte excretion were measured before and for 3 hours after drug intake on days 1 and 7. RESULTS: Celecoxib had no effect on systemic blood pressure, but short-term transient decreases in renal blood flow and glomerular filtration rate were found with the highest dose of 400 mg on day 1. On the first day, both celecoxib and naproxen decreased urine output (P < .05) and sodium, lithium, and potassium excretion (P < .01). On day 7, similar effects on water and sodium excretion were observed. During repeated administration, a significant sodium retention occurred during the first 3 days. CONCLUSION: In salt-depleted subjects, selective inhibition of COX-2 causes sodium and potassium retention. This suggests that an increased selectivity for COX-2 does not spare the kidney, at least during salt depletion.

In vivo targeting of an anti-tumor antibody coupled to antigenic MHC class I complexes induces specific growth inhibition and regression of established syngeneic tumor grafts.

The concept of antibody-mediated targeting of antigenic MHC/peptide complexes on tumor cells in order to sensitize them to T-lymphocyte cytotoxicity represents an attractive new immunotherapy strategy. In vitro experiments have shown that an antibody chemically conjugated or fused to monomeric MHC/peptide can be oligomerized on the surface of tumor cells, rendering them susceptible to efficient lysis by MHC-peptide restricted specific T-cell clones. However, this strategy has not yet been tested entirely in vivo in immunocompetent animals. To this aim, we took advantage of OT-1 mice which have a transgenic T-cell receptor specific for the ovalbumin (ova) immunodominant peptide (257-264) expressed in the context of the MHC class I H-2K(b). We prepared and characterized conjugates between the Fab' fragment from a high-affinity monoclonal antibody to carcinoembryonic antigen (CEA) and the H-2K(b) /ova peptide complex. First, we showed in OT-1 mice that the grafting and growth of a syngeneic colon carcinoma line transfected with CEA could be specifically inhibited by systemic injections of the conjugate. Next, using CEA transgenic C57BL/6 mice adoptively transferred with OT-1 spleen cells and immunized with ovalbumin, we demonstrated that systemic injections of the anti-CEA-H-2K(b) /ova conjugate could induce specific growth inhibition and regression of well-established, palpable subcutaneous grafts from the syngeneic CEA-transfected colon carcinoma line. These results, obtained in a well-characterized syngeneic carcinoma model, demonstrate that the antibody-MHC/peptide strategy can function in vivo. Further preclinical experimental studies, using an anti-viral T-cell response, will be performed before this new form of immunotherapy can be considered for clinical use.

Renal hemodynamic and natriuretic effects of concomitant Angiotensin-converting enzyme and neutral endopeptidase inhibition in men.

This double-blind placebo-controlled study was designed to investigate the acute and sustained hormonal, renal hemodynamic, and tubular effects of concomitant ACE and neutral endopeptidase (NEP) inhibition by omapatrilat, a vasopeptidase inhibitor, in men. Thirty-two normotensive subjects were randomized to receive a placebo, omapatrilat (40 or 80 mg), or the fosinopril/hydrochlorothiazide (FOS/HCTZ; 20 and 12.5 mg, respectively) fixed combination for 1 week. Blood pressure, renal hemodynamics, urinary electrolytes and atrial natriuretic peptide excretion, and several components of the renin-angiotensin system were measured for 6 hours on days 1 and 7 of drug administration. When compared with the placebo and the FOS/HCTZ combination, omapatrilat induced a significant decrease in plasma angiotensin II levels (P<0.001 versus placebo; P<0.05 versus FOS/HCTZ) and an increase in urinary atrial natriuretic peptide excretion (P<0.01). These hormonal effects were associated with a significant fall in blood pressure (P<0.01) and a marked renal vasodilatation, but with no significant changes in glomerular filtration rate. The FOS/HCTZ markedly increased urinary sodium excretion (P<0.001). The acute natriuretic response to FOS/HCTZ was significantly greater than that observed with omapatrilat (P<0.01). Over 1 week, however, the cumulative sodium excretion induced by both doses of omapatrilat (P<0.01 versus placebo) was at least as great as that induced by the dose of FOS/HCTZ (P=NS versus FOS/HCTZ). In conclusion, the results of the present study in normal subjects demonstrate that omapatrilat has favorable renal hemodynamic effects. Omapatrilat combines potent ACE inhibition with a sustained natriuresis, which explains its well-documented potent antihypertensive efficacy.

The C-Jun N-Terminal Kinase Inhibition in Intracerebral Hemorrhage

Background: In intracerebral hemorrhage (ICH), a subtype of stroke, the bloodentry into the brain triggers toxicity resulting in a strong loss of neurons andinflammation. Water content is also increases leading to growing intracranial pressure,which worsens neurological outcome. C-Jun N-terminal kinases (JNKs) areactivated in response to stress stimuli. Specific inhibition of JNK by a TAT-coupledpeptide (XG-102) mediates neuroprotection in several models of ischemic stroke.Recently, we have noted that the JNK pathway is also activated in a mouse modelof ICH, raising the question of the efficacy of XG-102 in this model.Method: ICH was induced in the mouse by intrastriatal injection of bacterialcollagenase (0,1U). Three hours later, animals received an i.v. injection of XG-102(100μg/kg). The neuroscore was assessed using a scale (from 0 to 9) based on 3behavioral tests performed daily. Then, mice were sacrificed at 6h, 24h, 48h and 5dafter ICH and histological studies performed.Results: XG-102 significantly improves neurological outcome at 24h (mean score:1,8±1.4 vs 3,4±1.8, p<0.01). Analysis of the lesion volume revealed a significantdecrease of the lesion area in the treated group at 48h (29±11 mm3 vs 39±5 mm3,p = 0.04). XG-102 mainly inhibits the edema component of the lesion. Indeed, asignificant decrease of the brain swelling was observed in treated animals at 48h(14±13% vs 26±9%, p=0.04) and 5d (-0,3±4.5% vs 5,1±3.6%, p=0.01).Conclusions: Inhibition of the JNK pathway by XG-102 appears to lead to asignificant decrease of the cerebral edema in the ICH model providing a furtherbeneficial effect of the XG-102 treatment. This result is of interest becausecurrently, clinical treatment for brain edema is limited. Importantly, the beneficialeffects observed with XG-102 in both stroke models open the possibility to rapidlytreat patients before identifying the stroke subtype by imaging.

The CAP1/Prss8 catalytic triad is not involved in PAR2 activation and protease nexin-1 (PN-1) inhibition.

Serine proteases, serine protease inhibitors, and protease-activated receptors (PARs) are responsible for several human skin disorders characterized by impaired epidermal permeability barrier function, desquamation, and inflammation. In this study, we addressed the consequences of a catalytically dead serine protease on epidermal homeostasis, the activation of PAR2 and the inhibition by the serine protease inhibitor nexin-1. The catalytically inactive serine protease CAP1/Prss8, when ectopically expressed in the mouse, retained the ability to induce skin disorders as well as its catalytically active counterpart (75%, n=81). Moreover, this phenotype was completely normalized in a PAR2-null background, indicating that the effects mediated by the catalytically inactive CAP1/Prss8 depend on PAR2 (95%, n=131). Finally, nexin-1 displayed analogous inhibitory capacity on both wild-type and inactive mutant CAP1/Prss8 in vitro and in vivo (64% n=151 vs. 89% n=109, respectively), indicating that the catalytic site of CAP1/Prss8 is dispensable for nexin-1 inhibition. Our results demonstrate a novel inhibitory interaction between CAP1/Prss8 and nexin-1, opening the search for specific CAP1/Prss8 antagonists that are independent of its catalytic activity.-Crisante, G., Battista, L., Iwaszkiewicz, J., Nesca, V., Mérillat, A.-M., Sergi, C., Zoete, V., Frateschi, S., Hummler, E. The CAP1/Prss8 catalytic triad is not involved in PAR2 activation and protease nexin-1 (PN-1) inhibition.

The dexamethasone-induced inhibition of proliferation, migration, and invasion in glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF) and can be enhanced by specific MIF inhibitors.

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.

Hemodynamic and biochemical consequences of renin inhibition by infusion of CGP 38560A in normal volunteers.

Hemodynamic and biochemical effects of the new renin inhibitor CGP 38560A (molecular weight 826) were tested in 15 healthy volunteers after a single-blind, randomized, placebo-controlled protocol. At a 2-week interval, groups of five subjects received a 30-minute infusion of either 5% dextrose or CGP 38560A 50, 125, or 250 micrograms/kg. Blood pressure, heart rate, plasma renin activity, active and total renin, angiotensin-(1-8)octapeptide (angiotensin II), and aldosterone were sequentially measured up to 3 hours from the onset of the infusion. There was no consistent change in blood pressure or heart rate. Plasma renin activity and angiotensin II decreased dose dependently, and peak suppression was observed at the end of the infusion of CGP 38560A and after the 250-micrograms/kg dose. Plasma renin activity fell from 1.0 +/- 0.19 (mean +/- SEM) to less than 0.05 ng/ml/hr in all five subjects (p less than 0.001), and angiotensin II fell from 7.7 +/- 1.2 to 2.6 +/- 0.9 femtomole/ml (p less than 0.01). Active renin rose fourfold from 24 +/- 1.9 to 98 +/- 14 pg/ml (p less than 0.001) at the end of the infusion of the high dose. Plasma angiotensin II returned toward its initial values much faster than plasma renin activity and active renin. In conclusion, CGP 38560A was well tolerated. It induced a dose-dependent decrease in angiotensin II and plasma renin activity and a long-lasting and dose-dependent rise in active renin. The doses used did not reduce plasma angiotensin II maximally despite reduction of plasma renin activity to unmeasurable levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition.

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

Pharmacologic profile of UR-7247, an orally active angiotensin II AT1 receptor antagonist, in healthy volunteers. p6.

This study was conducted to assess the pharmacologic properties of the new orally active angiotensin II subtype I (AT1) antagonist UR-7247, a product with a half-life >100 h in humans. The experiment was designed as an open-label, single-dose administration study with four parallel groups of four healthy men receiving increasing single oral doses (2.5, 5, and 10 mg) of UR-7247 or losartan, 100 mg. Angiotensin II receptor blockade was investigated < or =96 h after drug intake, with three independent methods [i.e., the inhibition of blood pressure (BP) response to exogenous Ang II, an in vitro Ang II-receptor assay (RRA), and the reactive increase in plasma angiotensin II. Plasma drug levels also were measured. The degree of blockade observed in vivo was statistically significant < or = 96 h with all UR-7247 doses for diastolic BP (p < 0.05) and < or =48 h for systolic BP. The maximal inhibition achieved with 10 mg UR-7247 was measured 6-24 h after drug intake and reached 54 +/- 17% and 48 +/- 20% for diastolic and systolic responses, respectively. Losartan, 100 mg, induced a greater short-term AT1-receptor blockade than 2.5- and 5.0-mg doses of UR-7247 (p < 0.001 for diastolic BP), but the UR-7247 effect was longer lasting. In vivo, no significant difference was observed between 10 mg UR-7247 and 100 mg losartan 4 h after drug intake, but in vitro, the blockade achieved with 100 mg losartan was higher than that seen with UR-7247. Finally, the results confirm that UR-7247 has a very long plasma elimination half-life, which may be due to a high but also tight binding to protein binding sites. In conclusion, UR-7247 is a long-lasting, well-tolerated AT1 receptor in healthy subjects.

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors.

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.