Thiostrepton-Nanomedicine, a TLR9 Inhibitor, Attenuates Sepsis-Induced Inflammation in Mice.

Sepsis is a life-threatening clinical condition caused by infection and transposition of pathogens and pathogen-associated molecular patterns (PAMPs) into the host bloodstream. During sepsis, activation of toll-like receptors (TLRs) on immune cells triggers the release of pro-inflammatory cytokines and overstimulates the production of vasodilatory mediators such as nitric oxide (NO). These vascular changes lead to widespread inflammation, tissue damage, multiple organ failure, and often death. New therapeutic options are urgently needed. To this end, thiostrepton (TST) has emerged as a candidate for sepsis treatment due to its action as an antibiotic and anti-inflammatory molecule (TLR7-9 inhibitor). Reports in the literature suggest that TLR9 inhibition substantially suppresses the excessive host inflammatory response and attenuates sepsis-induced mortality in the cecal ligation and puncture (CLP) murine model of sepsis. However, to the best of our knowledge, TST has never been directly tested as a therapeutic option for the management of sepsis, possibly due to its low water solubility and drug delivery issues. These facts prompted us to test the central hypothesis that TST encapsulated in phospholipid sterically stabilized micelles (TST-SSM) could be developed into a novel treatment for sepsis. Thus, using our published method of encapsulating the hydrophobic antibiotic TST-SSM, we evaluated the in vivo efficacy of TST-SSM nanomedicine in the murine model of polymicrobial sepsis. We found that TST-SSM increased the median survival of CLP-induced septic mice from 31 to 44 hr by reducing the bacterial burden in the blood and peritoneal lavage. Moreover, plasma levels of pro-inflammatory cytokines (interleukin 6 and tumor necrosis factor-alpha) and NO derivatives were also reduced, whereas renal and hepatic function biomarkers creatinine and aspartate transferase were significantly improved. In conclusion, we identified that TST-SSM nanomedicine has significant potential as a therapeutic agent for sepsis management, primarily due to its anti-inflammatory and antibiotic properties.

Insight into the beneficial immunomodulatory mechanism of the sevoflurane metabolite hexafluoro-2-propanol in a rat model of endotoxaemia.

Volatile anaesthetics such as sevoflurane attenuate inflammatory processes, thereby impacting patient outcome significantly. Their inhalative administration is, however, strictly limited to controlled environments such as operating theatres, and thus an intravenously injectable immunomodulatory drug would offer distinct advantages. As protective effects of volatile anaesthetics have been associated with the presence of trifluorinated carbon groups in their basic structure, in this study we investigated the water-soluble sevoflurane metabolite hexafluoro-2-propanol (HFIP) as a potential immunomodulatory drug in a rat model of endotoxic shock. Male Wistar rats were subjected to intravenous lipopolysaccharide (LPS) and thereafter were treated with HFIP. Plasma and tissue inflammatory mediators, neutrophil invasion, tissue damage and haemodynamic stability were the dedicated end-points. In an endotoxin-induced endothelial cell injury model, underlying mechanisms were elucidated using gene expression and gene reporter analyses. HFIP reduced the systemic inflammatory response significantly and decreased endotoxin-induced tissue damage. Additionally, the LPS-provoked drop in blood pressure of animals was resolved by HFIP treatment. Pathway analysis revealed that the observed attenuation of the inflammatory process was associated with reduced nuclear factor kappa B (NF-κΒ) activation and suppression of its dependent transcripts. Taken together, intravenous administration of HFIP exerts promising immunomodulatory effects in endotoxaemic rats. The possibility of intravenous administration would overcome limitations of volatile anaesthetics, and thus HFIP might therefore represent an interesting future drug candidate for states of severe inflammation.

Clinically relevant concentrations of lidocaine and ropivacaine inhibit TNFα-induced invasion of lung adenocarcinoma cells in vitro by blocking the activation of Akt and focal adhesion kinase.

BACKGROUND: Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells. METHODS: NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml(-1)) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed. RESULTS: Ropivacaine (1 nM-100 µM) and lidocaine (1-100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10(-6) M for lidocaine; IC50=1.52×10(-10) M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM). CONCLUSIONS: At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

Regional anaesthesia and cancer metastases: the implication of local anaesthetics.

Clinical and basic science studies have demonstrated the anti-inflammatory properties of local anaesthetics. Recent studies have begun to unravel molecular pathways linking inflammation and cancer. Regional anaesthesia is associated in some retrospective clinical studies with reduced risk of metastasis and increased long-term survival. The potential beneficial effects of regional anaesthesia have been attributed mainly to the inhibition of the neuroendocrine stress response to surgery and to the reduction in the requirements of volatile anaesthetics and opioids. Because cancer is linked to inflammation and local anaesthetics have anti-inflammatory effects, these agents may participate in reducing the risk of metastasis, but their mechanism of action is unknown. We demonstrated in vitro that amide local anaesthetics attenuate tumour cell migration as well as signalling pathways enhancing tumour growth and metastasis. This has provided the first evidence of a molecular mechanism by which regional anaesthesia might inhibit or reduce cancer metastases.

Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10µmol/L) and the Akt inhibitor V (20µmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway.

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G(i), and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial (125)I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid G(alphai) carboxyl-terminal peptide inhibited endothelial (125)I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of G(alphai) with the caveolin-1, whereas dn-Src inhibited G(alphai) binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream G(i)-coupled Src kinase signaling pathway.

Transport across the endothelium: regulation of endothelial permeability.

An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways controlling endothelial barrier function have been identified. The transcellular pathway transports plasma proteins of the size of albumin or greater via the process of transcytosis in vesicle carriers originating from cell surface caveolae. Specific signalling cues are able to induce the internalisation of caveolae and their movement to the basal side of the endothelium. Caveolin-1, the primary structural protein required for the formation of caveolae, is also important in regulating vesicle trafficking through the cell by controlling the activity and localisation of signalling molecules that mediate vesicle fission, endocytosis, fusion and finally exocytosis. An important function of the transcytotic pathways is to regulate the delivery of albumin and immunoglobulins, thereby controlling tissue oncotic pressure and host-defence. The paracellular pathway induced during inflammation is formed by gaps between endothelial cells at the level of adherens and tight junctional complexes. Paracellular permeability is increased by second messenger signalling pathways involving Ca2+ influx via activation of store-operated channels, protein kinase Calpha (PKCalpha), and Rho kinase that together participate in the stimulation of myosin light chain phosphorylation, actin-myosin contraction, and disruption of the junctions. In this review of the field, we discuss the current understanding of the signalling pathways regulating paracellular and transcellular endothelial permeability.

Bradykinin B2 receptors on skeletal muscle are coupled to inositol 1,4,5-trisphosphate formation.

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.

Thromboxane A2 mediates the stimulation of inositol 1,4,5-trisphosphate production and intracellular calcium mobilization by bradykinin in neonatal rat ventricular cardiomyocytes.

Bradykinin is a mediator of the protection of myocardium by angiotensin I-converting enzyme/kininase II inhibitors. We reported that the activation of B2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 response to 10 nmol/L bradykinin from 570 +/- 20.0 to 1150 +/- 51.3 and to 1016.7 +/- 21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.

Ovarian steroid protection against coronary artery hyperreactivity in rhesus monkeys.

Our hypothesis was that estrogen and progesterone modulate coronary artery reactivity in rhesus monkeys. Adult ovariectomized (ovx) monkeys were treated for 1, 2, or 4 wk with physiological concentrations of 17 beta-estradiol (E2), natural progesterone (P), and/or therapeutic levels of medroxyprogesterone acetate (MPA). Steroid concentrations in venous blood, coronary artery estrogen receptor (ER) and progesterone receptor (PR) localization, and isolated vascular muscle cell (VMC) Ca2+ and protein kinase C responses to serotonin and U46619 (a thromboxane A2 mimetic) were measured. Ovx monkey VMC responses were hyperreactive, showing prolonged increases in intracellular Ca2+ and protein kinase C that correlated with exaggerated in vivo coronary artery vasoconstrictor responses. The hyperreactive Ca2+ responses were abolished by in vivo treatment with E2 and/or P. However, VMC from ovx monkeys treated with the combination of E2 and MPA or E2, P, and MPA remained hyperreactive to vasoconstrictor stimuli, suggesting that MPA negated the protective effects of E2. ER were detected primarily in interstitial and endothelial cells and a minor fraction of the VMC. PR were localized to coronary artery VMC and interstitial cell nuclei. In vivo treatment of ovx monkeys with E2 tended to up-regulate PR in VMC, but MPA appeared to down-regulate PR expression. These results suggest that E2 and P replacement decreases coronary artery reactivity through direct interactions with ER and PR in coronary artery VMC.

Angiotensin I-converting enzyme inhibitors potentiate bradykinin's inotropic effects independently of blocking its inactivation.

The positive inotropic effects of bradykinin (BK) and 2 analogs resistant to angiotensin I-converting enzyme (ACE) were potentiated on isolated guinea pig atrial preparations by enalaprilat. The stable BK analogs, dextran-BK and [Hyp3-Tyr(Me)8]-BK, were as active as BK. Pretreatment for 5 min with enalaprilat augmented the maximal positive inotropic effect of [Hyp3-Tyr(Me)8]-BK 2.8-fold, from 19% to 53% and that of BK from 28% to 42% over baseline; inotropic responses to dextran-BK (1 microM) were similarly increased. The activity of atrial ACE, a zinc-requiring enzyme, was completely inhibited by 8-hydroxyquinoline-5-sulfonic acid (QSA, 10 mM), which raised the maximal inotropic effect of BK to 39% above baseline. This value rose to 67% when in addition to QSA, 1 microM enalaprilat was added; enalaprilat thus, potentiated the effects of BK independently of enzyme inhibition. The positive inotropic effects to BK and its analogs decline with time in the presence of these agonists. After 10 min of exposure, the response to 1 microM [Hyp3-Tyr(Me)8]-BK decreased to about half, and after 20 min, to 0. Enalaprilat, when present in the tissue bath, prevented the decline in inotropy; even after tachyphylaxis occurred, it reversed this decrease in activity when added. The effects of 1 microM [Hyp3-Tyr(Me)8]-BK, in the absence or presence of enalaprilat, were abolished by the BK B2 receptor antagonist icatibant (0.75 microM). The results indicate that ACE inhibitors, by potentiating the BK effects and blocking BK B2-receptor desensitization, may contribute to the beneficial cardiac effects of BK independently of blocking its inactivation.

Are the inotropic and antiarrhythmic effects of bradykinin due to increases in coronary flow?

The purpose of this research was to test whether the positive inotropic and antiarrhythmic effects of bradykinin are due solely to increases in coronary flow. Rat hearts were perfused at constant pressure (75 cm H2O) and temperature (37 degrees C). Coronary flow was measured using an electronic drop counter. Contractile force was assessed using a left ventricular balloon catheter. Bradykinin (10 nmol/L) significantly increased coronary flow by 55 +/- 8% above the control level of 4.8 +/- 0.5 mL/min (n = 20), while force was increased by 23.1 +/- 3% (n = 20). Ramiprilat (10 nmol/L) potentiated the vasodilatory and inotropic responses to 10 nmol/L bradykinin by 58 +/- 8% (n = 5). When hearts were perfused at constant flow, bradykinin no longer produced a positive inotropic effect. Bradykinin, 10 or 100 nmol/L, under these conditions actually caused a negative inotropic effect of -24.8 +/- 5% (n = 8) and -35 +/- 11% (n = 3), respectively. In another 2 groups of hearts, also perfused at constant pressure, reperfusion arrhythmias were elicited after a 20-min period of complete global ischemia. In control hearts, the mean period of fibrillation was 7.3 +/- 1.8 min (n = 10). This period was significantly reduced to 2.7 +/- 0.7 min (n = 10) in hearts receiving 10 nmol/L bradykinin. In untreated hearts, the coronary flow during the reperfusion period increased over the baseline flow by a factor of 1.8 +/- 0.2, and this factor was not significantly effected by bradykinin. These results suggest that only the positive inotropic, but not the antiarrhythmic, action of bradykinin is due to coronary vasodilation.

Potentiation of the effects of bradykinin on its receptor in the isolated guinea pig ileum.

Angiotensin I-converting enzyme (ACE/kininase II) inhibitors potentiated guinea pig ileum's isotonic contractions to bradykinin (BK) and its analogues, shifting the BK dose-response curve to the left. ACE inhibitors added at the peak of the contraction immediately enhanced it further (343 +/- 40%), although the ileum inactivated BK slowly (t(1/2) = 12-16 min). Chymotrypsin and cathepsin G also augmented the activity of BK up to three- or four-fold, but in a manner slower than that of ACE inhibitors. The BK B(2) receptor blocker HOE 140 inhibited all effects. Histamine and angiotensin II were not potentiated. ACE inhibitors potentiate BK independent of blocking its inactivation by inducing crosstalk between ACE and the BK B(2) receptor; proteases activate the receptor by different mechanism.

Lack of potent antinociceptive activity by substance P antagonist CP-96,345 in the rat spinal cord.

Substance P (SP) binds to the NK-1 receptor and has been implicated in the transmission of pain as well as in physiological responses such as salivary gland secretion and neurogenic inflammation. Studies in this field have been limited due to the lack of specific antagonists that are not degraded rapidly and are not neurotoxic. However, a recently developed non-peptide SP antagonist, CP-96,345, is specific for the NK-1 receptor. The purpose of this study was to assess the effects of this antagonist on nociception. The tail flick, paw pinch and hot plate tests were used to assess nociception in rats. Following baseline determination of tail skin temperature and analgesiometric tests, the rats received intrathecal injections of various doses of CP-96,345, and pain sensitivity was assessed at several time intervals up to two hours after injection. The ability of CP-96,345 to inhibit SP induced biting and scratching was also assessed. Results from the analgesiometric tests indicated that there were no significant elevations in the latency of tail flick test or the paw pinch thresholds even at 240 micrograms of CP-96,345. The hot plate latency was elevated at the highest dose of antagonist. In addition, there was a significant dose-related elevation in latency on the hot plate test. CP-96,345 also produced a dose-related decrease in tail skin temperature. CP-96,345 did not block SP induced biting and scratching. CP-96,345 and SP were evaluated for their ability to displace 125I-Tyr8-SP from rat spinal cord, brain and submandibular gland membrane fractions. It was found that although the affinity of CP-96,345 was 56 fold lower than that of SP in the brain, the antagonist was nearly as potent as SP in the spinal cord and submandibular gland. The results of this study suggest that, while CP-96,345 binds to the NK-1 receptor in the spinal cord, this receptor is most likely not involved in mediating some types of nociception at the spinal cord level.

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation.

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.

Interaction of insulin-like growth factor I with turkey satellite cells and satellite cell-derived myotubes.

Satellite cells, isolated from the superficial pectoralis muscle of growing Nicholas tom turkeys, were cloned to obtain a pure population of myogenic cells. These cells proliferated rapidly and differentiated (fused) into myotubes typically containing 92-98% fused nuclei. Competitive binding assays were performed on near-confluent satellite cell or myotube cultures in 35 mm diameter wells by adding [125I]IGF-I along with increasing concentrations of unlabeled IGF-I, IGF-II, or insulin. Following incubation, the cultures were washed to remove the unbound hormones, solubilized with 0.5 N NaOH, and the radioactivity specifically bound was determined. Total and fused nuclei number as well as total protein were determined in parallel cultures. Our results indicate that turkey satellite cell and myotube cultures possess specific binding sites for IGF-I. Displacement of [125I]IGF-I was in the order of IGF-I greater than IGF-II greater than or equal to insulin. Although the [125I]IGF-I association constants were similar for turkey satellite cells and myotubes, a 2.8-fold decrease in the number of receptors per nuclei was observed as satellite cells differentiated into myotubes. The 50% inhibition constants for IGF-I, IGF-II, and insulin were 3.7 X 10(-9) M, 7.5 X 10(-8) M, and 8.7 X 10(-8) M for satellite cells and 3.1 X 10(-9) M, 7.5 X 10(-8) M, and 9.6 X 10(-8) M for myotubes, respectively. Receptor cross-linking analysis using disuccinimidyl suberate was performed on near-confluent satellite cell cultures incubated with [125I]IGF-I in the presence or absence of 1 X 10(-7) M IGF-I, IGF-II, or insulin. Receptor subunit species of Mr 130 kDa and 98 kDa were observed under reducing conditions (100 mM dithiothreitol) and at a Mr greater than 300 kDa (native receptor tetramer) under non-reduced conditions. Autoradiographic bands were displaced with IGF-I but not with equimolar levels of IGF-II or insulin. The results suggest that turkey satellite cells possess a type I IGF receptor.

Satellite cells of growing turkeys: influence of donor age and sex on proliferation and differentiation in vitro.

Proliferation and differentiation (fusion) of myogenic satellite cells isolated from Nicholas tom and hen turkeys at 3, 9, and 15 weeks of age and the response of a satellite cell clone to serum from these birds were examined. Responsiveness of satellite cells to culture conditions was proportional to donor age, and sex of bird had no effect on satellite cell proliferation. Fusion percentages were similar in all cultures. When a turkey satellite cell clone was exposed to serum from tom and hen turkeys at each age, an age-related decline in proliferation occurred with serum from hen but not from tom turkeys. Minimal cell fusion occurred in turkey serum and neither proliferation nor fusion was correlated with serum IGF-I levels. Cells derived from pectoralis major and anterior latissimus dorsi muscles of 15-week-old tom turkeys possessed similar proliferation and fusion properties in vitro. It appears that satellite cells retain a high proliferative activity throughout growth in turkeys, and the mitogenic properties of turkey serum may be evaluated using turkey satellite cells in culture.

The influence of chronic hypokalemia on myocardial adrenergic receptor densities: enhanced sensitivity to epinephrine-induced arrhythmias.

We studied the effects of a 30-day potassium (K+)-deficient diet on blood [K+] myocardial adrenergic receptor densities, serum catecholamines, and epinephrine arrhythmogenicity in adult laboratory rats (250 +/- 25 g). Within 3 days of beginning the K+-deficient diet, blood [K+] decreased by 50%. After 5 days, the myocardial alpha-1 density increased (62 +/- 2 vs 148 +/- 16 fmols/mg protein), and the total beta receptor increased (95 +/- 5 vs 273 +/- 49) without significant change in receptor affinity. However, 18-21 days of this diet was necessary to produce an increase in the duration of epinephrine arrhythmias (from 56 +/- 8 to 224 +/- 21 s). While prazosin block of the alpha-1 receptor in hypokalemic rats caused a significant, 42% reduction in arrhythmic duration and propranolol block caused a 62% reduction, both prazosin and propranolol were necessary to return arrhythmia times to normal (44 +/- 0.3 mmols/dL). Total serum catecholamines were reduced after 3 days of the diet (from 482 +/- 37 to 299 +/- 31 pg/ml) and remained depressed throughout the 30 days of the K+ diet. The results of this study indicate that prolonged restriction causes a reduction in serum catecholamines, an increase in myocardial alpha-1 and beta receptors densities, and an increase in epinephrine arrhythmogenicity. All of these changes were reversed within 5 days of initiating a normal dietary K+ intake.

beta(2)-Integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice.

Interaction of CD11/CD18 beta(2) integrins on polymorphonuclear leukocytes (PMNs) with their counterreceptor, intercellular adhesion molecule-1, on the surface of vascular endothelial cells is a critical event mediating stable PMN adhesion and migration across the pulmonary vascular endothelial barrier. Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated from the canine hookworm (Ancylostoma caninum), binds to the I domain of CD11a and CD11b and inhibits beta(2) integrin-dependent PMN adhesion. We describe a novel strategy using the endothelial cell-specific E-selectin promoter to induce NIF expression in an inflammation-specific manner in pulmonary vascular endothelial cells. A construct containing NIF cDNA driven by the inducible endothelial cell-specific E-selectin promoter (pESNIF) was transfected into human pulmonary artery endothelial cells (HPAECs). Lipopolysaccharide challenge (known to activate E-selectin) resulted in NIF mRNA and protein expression in transfected HPAECs. NIF expression induced by the E-selectin promoter prevented PMN adhesion to the activated HPAECs, whereas PMNs adhered avidly to activated HPAECs in the absence of NIF expression. To address the utility of this approach in conditionally preventing in vivo PMN sequestration, we injected mice intravenously with cationic liposomes containing the pESNIF construct. Analysis of lung tissue showed that intraperitoneal challenge of Escherichia coli resulted in NIF expression. Inflammation-specific NIF expression induced by the E-selectin promoter prevented lung PMN sequestration and vascular injury induced by E coli challenge. These studies suggest the feasibility of conditionally blocking beta(2) integrin function at sites where the endothelium is activated and thereby of locally preventing PMN activation and migration responses that lead to tissue inflammation.

Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor.

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346. RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b. However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.