A-type K+ channels contribute to the prorenin increase of firing activity in hypothalamic vasopressin neurosecretory neurons.

Recent studies have supported an important contribution of prorenin (PR) and its receptor (PRR) to the regulation of hypothalamic, sympathetic, and neurosecretory outflows to the cardiovascular system, including systemic release of vasopressin (VP), both under physiological and cardiovascular disease conditions. Still, the identification of precise cellular mechanisms and neuronal/molecular targets remain unknown. We have recently shown that PRR is expressed in VP neurons and that their activation increases neuronal activity. However, the underlying ionic channel mechanisms are undefined. Here, we performed patch-clamp electrophysiology from identified VP neurons in acute hypothalamic slices obtained from enhanced green fluorescent protein-VP transgenic rats. Voltage-clamp recordings showed that PR inhibited the magnitude of A-type K+ current (IA; ~50% at -25 mV), a subthreshold voltage-dependent current that restrains VP firing activity. PR also increased the inactivation rate of IA and shifted the steady-state voltage-dependent inactivation function toward more hyperpolarized membrane potential (~7 mV shift), thus resulting in less channel availability to be activated at any given membrane potential. PR also inhibited a sustained component of IA ("window" current). PR-mediated changes in action potential waveform and increased firing activity were occluded when IA was blocked by 4-aminopyridine. Finally, PR failed to increase superoxide production within the supraoptic nucleus/paraventricular nucleus, and PR excitatory effects persisted in slices treated with the SOD mimetic tempol. Taken together, these experiments indicated that PR excitatory effects on vasopressin neurons involve inhibition of IA, due, in part, to increases in its voltage-dependent inactivation properties. Moreover, our results indicate that PR effects did not involve an increase in oxidative stress.NEW & NOTEWORTHY Here, we demonstrate that prorenin/the prorenin receptor is an important signaling unit for the regulation of vasopressin firing activity and, thus, systemic hormonal release. We identified A-type K+ channels as key molecular targets mediating prorenin stimulation of vasopressin neuronal activity, thus standing as a potential therapeutic target for neurohumoral activation in cardiovascular disease.

Differential effects of serine proteases on the migration of normal and tumor cells: implications for tumor microenvironment.

The supporting role of proteases in tumor progression and invasion is well known; however, the use of proteases as therapeutic agents has also been demonstrated. In this article, the authors report on the differential effects of exogenous serine proteases on the motility of tumor and normal cells. The treatment of normal and tumor cells with a single dose of pancreatic serine proteases, trypsin (TR) and chymotrypsin (CH), leads to a concentration-dependent response by cells, first accelerating and then slowing mobility. Tumor cells are 10 to 20 times more sensitive to exogenous TR/CH, suggesting that a single dose of proteases may cause discordant movements of normal and tumor cells within the tumor environment. The inhibitory effects of TR on cell motility are contradicted by thrombin (TH), particularly in the regulation of normal cells' migration. The purpose of this investigation was to ascertain the role of protease-activated receptors (PARs) in terms of normal and tumor cell motility. Duplicate treatments with proteases resulted in diminished mobility of both normal and tumor cells. Repeated application of TR and TH in 1-hour treatment intervals initially desensitizes cell surface PARs. However, cell surface PARs reappear regardless of subsequent protease treatments in both normal and tumor cells. The resensitization process is retarded in tumor cells when compared with normal cells. This is evidenced by lower expression of PARs as well as by their relocalization at the tumor cell surfaces. Under these conditions, normal cells remain responsive to exogenous proteases in terms of cell motility. Exogenous proteases do not modulate motility of repeatedly stimulated tumor cells, and consequently, the migration of tumor cells appears disconnected from the PAR signaling pathways. The use of activating peptides in lieu of the cognate proteases for a given PAR system indicated that proteases may act through additional targets not regulated by PAR signaling. We hypothesize that the divergent migration patterns of normal and tumor cells due to exposure to proteases is in part mediated by PARs. Thus, treatment with exogenous proteases may cause rearrangement of the tumor and stromal cells within the tumor microenvironment. Such topographical effects may lead to the inhibition of tumor progression and metastasis development.

A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia.

Neuronal cell death occurs during many neurodegenerative disorders and stroke. The aberrant, excessive activity of matrix metalloproteinases (MMPs), especially MMP-9, contributes directly to neuron apoptosis and brain damage (Rosenberg et al., 1996; Asahi et al., 2001; Gu et al., 2002; Horstmann et al., 2003). We determined that MMP-9 degrades the extracellular matrix protein laminin and that this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice. We also determined that the highly specific thiirane gelatinase inhibitor SB-3CT blocks MMP-9 activity, including MMP-9-mediated laminin cleavage, thus rescuing neurons from apoptosis. We conclude that MMP-9 is a highly promising drug target and that SB-3CT derivatives have significant therapeutic potential in stroke patients.

Effects of dopaminergic drugs on the mast cell degranulation and nitric oxide generation in RAW 264.7 cells.

Effects of dopaminergic drugs on the degranulation of mast cells (RBL-2H3 cells) and the nitric oxide production from macrophage cells (RAW 264.7) were studied. Among the dopaminergic agonists and antagonists tested, bromocriptine, 7-OH-DPAT, haloperidol, and clozapine showed potent inhibitions of mast cell degranualtion (IC50 value, 5 microM). However, these dopaminergic agents did not affect the tyrosine phosphorylations of the signaling components of the high affinity IgE receptor (FcepsilonRI), such as Syk, PLCgamma1, and PLCgamma2.; This suggested that these signaling components were not involved in the inhibition of the mast cell degranulation by these compounds. On the other hand, dopamine, bromocriptine, 7-OH-DAPT, and haloperidol markedly inhibited the nitric oxide production from RAW 264.7 cells (IC50 values, 10-20 microM). Bromocriptine, a dopamine agonist that is routinely used for the treatment of Parkinsons disease, inhibited the expression of the inducible nitric oxide synthase at an early stage of the LPS-induced protein expression in a dose-dependent manner. The results suggested that these dopaminergic agents, when used for the treatment of dopamine receptors-related diseases, such as Schizophrenia or Parkinsons disease, might have additional beneficial effects.

Effects of moxifloxacin in zymogen A or S. aureus stimulated human THP-1 monocytes on the inflammatory process and the spread of infection.

Antimicrobial agents have been reported to exhibit immunomodulatory and anti-inflammatory activities, both in vivo and in vitro (e.g., in human lymphocytes, macrophages and monocytes). The effects of moxifloxacin on cytokine immunomodulatory mediators, free radical generation and hydrolytic enzyme activities in zymogen A-stimulated human THP-1 monocytes were evaluated. An increase in c-AMP levels, protein kinase C activity, and the release of nitric oxide and hydrogen peroxide with a decrease in pH occurred within the first hour. Further, the effects of moxifloxacin were reduced by agents which blocked the oxygen burst, lysosome-phagosome fusion, and the energy generation within the cell. After 4 h, there was a decrease in NAG and cathepsin D activities, lipid peroxidation and the release of pro-inflammatory cytokines. These data indicate that moxifloxacin may modify the acute-phase inflammatory responses through inhibition of cytokine release in monocytes. Moxifloxacin inhibited the release of TNFalpha, IL-1, IL-6, and IL-8 in a concentration-dependent manner across a range of 0.004 to 4 microg/mL. After 4 h, there was a decrease in the release of these cytokines, thus interfering with the inflammation process to reduce infection and its spread. The effects of moxifloxacin appear initially to activate monocytes to kill bacteria through the innate immune process by releasing ROS and lysosomal hydrolytic enzymes as well as phagocytosis of the organism. At a later time the bacteria are killed through a Bacterialstatic mechanism of protein synthesis inhibition and there is a reversal of the effects of moxifloxacin on cytokine release, free radical generation and hydrolytic enzymes so that lipid peroxidation and tissue destruction by the infection process is suppressed.

In-vitro anti-inflammatory and immunomodulatory effects of grepafloxacin in zymogen A- or Staphylococcus aureus-stimulated human THP-1 monocytes.

The effects of grepafloxacin on the release of cytokines, chemical mediators, hydrolytic enzyme activities, and lipoxygenation in zymogen A- or Staphylococcus aureus-stimulated human THP-1 monocytes were evaluated. Initially, consistent with stimulation of phagocytic mechanisms of the monocytes, increases in cyclic adenosine monophosphate (cAMP) release, nitric oxide [NO] release, and hydrogen peroxide [H(2)O(2)] release, with a small decrease in cellular pH, occurred within 2 h. Enzymatic activities associated with oxygen burst of phagocytic cells (e.g., protein kinase C and nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase) were elevated, suggesting that monocytes attempted to destroy the invading organism through an innate phagocytic cidal immunologic mechanism. After 1-2 h of exposure to grepafloxacin, the oxygen burst and the release of proinflammatory cytokines and chemical mediators were suppressed. After 4 h, suppression of n-acetyl glucosaminidase (NAG) and cathepsin D activities and lipid peroxidation occurred, suppressing the pathogen-induced spread of infection and inflammation. Release of tumor necrosis factor (TNFalpha), interleukin (IL)-1, IL-6, and IL-8 was inhibited by grepafloxacin in a concentration-dependent manner, suggesting a reduction in the acute-phase inflammatory responses initiated by cytokine release from monocytes. Later, S. aureus were killed through inhibition of DNA synthesis, consistent with a bacteriostatic effect. Drug action against invading organisms appears to occur through multiple processes. Modulation of the innate immune system occurs within the first hour, causing the activation of cytokines, chemical mediators, and hydrolytic enzymes. A second phase between 2-4 h appears to involve the suppression of cellular components involved in inflammation and the spread of the infection. The third response, an apparent bacteriostatic inhibition of DNA synthesis, causes bacterial death.

Urocortin promotes hemodynamic and bioenergetic recovery and improves cell survival in the isolated rat heart exposed to ischemia/reperfusion.

OBJECTIVES: This study evaluates the hemodynamic, bioenergetic and cytoprotective effects of urocortin (Ucn) in the isolated rat heart exposed to ischemia (I)/reperfusion (R). BACKGROUND: We have previously demonstrated that administration of exogenous Ucn reduces infarct size in ischemic-reperfused rat hearts. METHODS: Urocortin 10(-8)M was added to the perfusate before I, before I and during R, and during R alone in the isolated pulsed rat heart exposed to 35 min I followed by 60 min R. RESULTS: Partial to complete recovery of diastolic pressure and developed pressure was seen irrespective of when Ucn was perfused. In particular, beneficial effects are observed when Ucn is only given during R. Urocortin given only before I, and before I and over R, although not during R alone, also produces significant recovery of high-energy phosphate pools. In each group, improvement in ventricular function is associated with reduction both in myocardial damage, assessed by creatine phosphokinase release, and in endothelial cell and cardiomyocyte apoptosis, assessed by caspase 3 activity and fluorescent-based terminal deoxynucleotidyl transferase mediated nick end labelling enhanced with counterstains. These improvements in ventricular performance, bioenergetics and cell survival are not secondary to any inotropic effects of Ucn. CONCLUSIONS: This is the first report to show enhanced cardiac function induced by Ucn during I/R. Because the cytoprotective and functional benefits are still produced when Ucn is given only at R, these data suggest that Ucn may be useful clinically in the management of myocardial infarction.

High therapeutic index of factor C Sushi peptides: potent antimicrobials against Pseudomonas aeruginosa.

Factor C protein isolated from the horseshoe crab, Carcinoscorpius rotundicauda, has endotoxin binding capability. Synthetic peptides of 34 amino acids based on the sequence of two regions of factor C (Sushi 1 and Sushi 3) as well as their corresponding mutants exhibited activities against 30 clinical isolates of Pseudomonas aeruginosa. Collectively, all four peptides demonstrated exceptionally effective bactericidal activity against P. aeruginosa with 90% minimal bactericidal concentrations (MBC(90)s) in the range of 0.06 to 0.25 microg/ml (16 to 63 nM). Viable bacteria were reduced by 90% after 7 min and were totally eradicated within 40 to 50 min. These peptides are minimally hemolytic against both rabbit and human erythrocytes even at concentrations up to 1,600-fold their MBC(90)s. Both in vitro and in vivo studies indicate that cytotoxic effects are small even at 1,000-fold their MBC(90)s. Furthermore, the Sushi peptides are tolerant of high-salt and adverse pH conditions. These findings demonstrate the promising therapeutic potential of the Sushi peptides.

New approaches to thrombolytic therapy.

Tissue-type plasminogen activator (t-PA), purified from the culture fluid of a stable human melanoma cell line, is a serine protease, different from urokinase, with a molecular weight of about 70,000. It is composed of one polypeptide chain, which is converted to a two-chain molecule by limited plasmic action. Activation of plasminogen to plasmin occurs by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis has shown that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate by increasing the affinity of plasminogen for fibrin-bound t-PA. The directed action of plasmin toward fibrin in vivo, might be explained by the low Michaelis constant in the presence of fibrin (0.16 microM), which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface would then be protected from rapid inactivation by alpha 2-antiplasmin. An important consequence of this molecular model for physiological fibrinolysis is that specific thrombolysis is only expected with the use of a specific plasminogen activator, which confines activation to the fibrin surface. Studies on the thrombolytic properties of purified t-PA in various animal species and in humans have revealed a higher specific thrombolytic activity than urokinase. Thrombolysis could be achieved without causing significant plasminogen activation, alpha 2-antiplasmin consumption, or fibrinogen breakdown. Alternatively, pro-urokinase, the zymogen precursor of urokinase, also displays a certain degree of fibrin specificity. Its mechanism of action and potential therapeutic value remain to be established.

[Conditions for immobilizing protosubtilin on aluminum oxide using transitional metal ions]. / Issledovanie uslovii immobilizatsii protosubtilina na okisi aliuminiia pri pomoshchi ionov perekhodnykh metallov.

The effect of the degree of the carrier activation by titanium tetrachloride, type of buffer solution, and protein concentration on characteristics of protosubtiline G10x immobilized on alumina was studied. X-ray phase analysis and IR spectroscopy were used to investigate the structure of the carrier and its changes during activation. To obtain the immobilized enzyme preparation with the maximum activity, immobilization should be carried out in 0.02 M Ca-acetate buffer on alumina treated with 2% solution of titanium tetrachloride.