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PubMed2004

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process induced by IAA in a NO-mediated but cGMP-independent pathway. The activation of MAPKs is discussed in relation to the cell responses modulating mitotic process.

Cucumis sativus↗
PubMed1977

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.

Animals↗
PubMed2002

Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins.

Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements obtained from FRET analysis. Following modification, stoichiometrically labeled cytochrome c was digested with trypsin and derivatized fragments were analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry to identify Lys25 as the predominant site of covalent modification. FRET analysis on derivatized protein demonstrated nearly complete quenching of Cascade Blue fluorescence, indicating the labeled lysine residue to reside within 30 A of the heme prosthetic group. Sodium dodecyl sulfate (SDS) denaturation resulted in an approximately 28% recovery of fluorescence, demonstrating the utility of this donor-acceptor pair for evaluating distance changes of 30-90 A. Modeling the Cascade Blue donor molecule onto Lys25 of a cytochrome c NMR structure confirmed a distance of < or =30 A from the heme acceptor, as determined by FRET analysis. Further modeling of the SDS-denatured state as an extended chain suggested a maximum separation distance of 45 A, also consistent with results derived from FRET analysis.

Animals↗
PubMed1994

Relationship between nitric oxide and vasoactive intestinal polypeptide in enteric inhibitory neurotransmission.

Although considerable evidence suggests that NO serves as a neurotransmitter in gastrointestinal muscles, it is unlikely to be the only substance involved in enteric inhibitory neurotransmission. Vasoactive intestinal polypeptide (VIP) is known to be expressed by inhibitory motor neurons in the gut, and it appears to be co-localized with nitric oxide synthase (NOS) in a subpopulation of enteric neurons. These data suggest that NO and VIP may be parallel neurotransmitters. Others have suggested that VIP is the primary inhibitory transmitter, and it stimulates production of NO in smooth muscle cells. In this "serial cascade" model NO is a paracrine substance. We performed experiments on circular muscles and cells from the canine proximal colon to further test the idea that NO and VIP are parallel neurotransmitters and to determine the validity of the serial cascade model in these muscles. We found that NO-independent inhibitory effects were unmasked when excitatory and NO-dependent inhibitory responses were blocked. NO-independent inhibitory effects were reduced by alpha-chymotrypsin and blocked by tetrodotoxin. NOS- and VIP-like immunoreactivities were co-localized in enteric neurons and varicose fibers in the circular muscle layer. Similar to several other reports we found no evidence for a constitutive NOS in smooth muscle cells. Several aspects of the serial cascade model were not supported by our results: (i) the electrical and mechanical effects of VIP did not depend upon NO synthesis; (ii) VIP-induced changes in [Ca2+]i did not depend upon NO synthesis; and (iii) VIP did not cause the release of NO from canine colonic muscles. These results are consistent with the hypothesis that NO and VIP are co-transmitters, released in parallel from enteric inhibitory nerves.

Animals↗
PubMed1998

Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR).

We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G1-to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40-75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3'-ends of DNA. Overnight pretreatment with 0.5-5.0 microM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B1, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25-100 microM fumonisin B1 resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide.

Anticarcinogenic Agents↗
PubMed1987

Thrombin stimulates arachidonate metabolism in murine tumor cells.

Thrombin can be formed in the tumor cell microenvironment following activation of the clotting cascade by procoagulants of cancer or host cells. We have tested here the effects of thrombin, either "endogenous" or "exogenous" (see below), on arachidonate mobilization from membrane phospholipids of mouse mammary tumor virus-induced (MMTV) carcinoma cells. These tumor cells exhibit in vitro a tissue type procoagulant activity (130 thromboplastin units/10(4) cells) and are therefore able to induce thrombin formation in a plasmatic milieu. To verify the effect of thrombin formation by tumor cell procoagulant ("endogenous thrombin"), either human or mouse platelet-free plasma (20% in DMEM) was added to the cell layer (prelabelled for 5 hr with a trace amount (0.013 microM) of 3H-arachidonate) and the system was recalcified (15 mM CaCl2). Thin-layer radiochromatography of the culture medium showed a significant release of 3H-labelled arachidonate products PGE2, PGF2 alpha and 6-ketoPGF1 alpha after 1 hr of incubation. To verify the effect of thrombin formation from host sources ("exogenous thrombin"), either bovine or purified human alpha-thrombin (0.1-10 U/ml) was added to the cells for different periods (from 5 min to 20 hr). Exogenous thrombin stimulated arachidonate release and metabolism in a dose-related manner. With short labelling periods (0.013 microM 3H-arachidonate for 30 min-1 hr) thrombin stimulated the release of unmetabolized 3H-arachidonate, but not of 3H-arachidonate metabolites. These processes were inhibited by a specific inhibitor of thrombin enzymatic activity (alpha-NAPAP, 140 microM) and by a cyclo-oxygenase inhibitor (ASA 4mM). Tumor-associated procoagulants may thus contribute not only to fibrin deposition but also to generation of multipotent mediators such as arachidonate metabolites.

Animals↗
PubMed2006

A test of three alternative pathways for consumer regulation of primary productivity.

The pathways linking consumer effects to primary productivity (PPR) are likely to vary among taxa because of species-specific trophic and functional differences. Thus, it is necessary to understand the dynamics of consumer-PPR interactions so that effects of species loss on ecosystem function can be addressed from a mechanistic approach. In this study, I used three fish taxa (orangethroat darter, Etheostoma spectabile; western mosquitofish, Gambusia affinis; and bullhead minnow, Pimephales vigilax) as model consumers with different trophic and functional characteristics to test alternative mechanisms for consumer regulation of PPR (i.e., trophic cascade, terrestrial nutrient translocation, and sedimentary nutrient translocation). Experiments were conducted in stream mesocosms fitted with a combination of fish and terrestrial insect barriers to address relative importance of consumer-driven top-down and bottom-up control of PPR. A predatory invertivore, orangethroat darter, increased PPR through an apparent trophic cascade by localized reduction of benthic grazing invertebrate densities (i.e., top-down). A surface feeding insectivore, western mosquitofish, consumed terrestrial insects on the stream surface, increasing PPR by enhancing allochthonous nutrients in the mesocosms (i.e., bottom-up). A benthic omnivore, bullhead minnow, consumed benthic food items, resulting in increased PPR by enhancing availability of autochthonous nutrients via translocation of sedimentary nutrients (i.e., bottom-up). However, under specific environmental contexts, this species also consumed terrestrial invertebrates, potentially affecting PPR through terrestrial nutrient translocation as well. In this study, the trophic and functional characteristics of different species resulted in alternative pathways that increased PPR, suggesting that in natural ecosystems multiple consumer-driven pathways may be influencing PPR simultaneously and could potentially be important for temporal persistence of ecosystem function in changing environments.

Analysis of Variance↗
PubMed1994

Effects of aprotinin on acute recovery of cerebral metabolism in piglets after hypothermic circulatory arrest.

Brain protection during cardiopulmonary bypass and hypothermic circulatory arrest is incomplete. Activation of blood protease cascades may contribute to cellular injury under these conditions. To test this hypothesis, effects of the protease inhibitor aprotinin on recovery of brain energy metabolism after hypothermic circulatory arrest were studied in the piglet. Twenty-four 4-week-old piglets (10 aprotinin-treated and 14 control) underwent core cooling, 1 hour of circulatory arrest at 15 degrees C, reperfusion and rewarming (45 minutes), and normothermic perfusion (3 hours) on cardiopulmonary bypass. Cerebral high-energy phosphate concentration and intracellular pH were studied by phosphorus-31 magnetic resonance spectroscopy in 12 animals. In the remaining animals cerebral and regional blood flow were measured with radioactive microspheres and carotid artery blood flow was measured with an electromagnetic flowmeter. Cerebral oxygen and glucose extraction were measured, and vascular resistance responses to endothelium-dependent (acetylcholine) and -independent (nitroglycerin) vasodilators were calculated. Recovery of cerebral adenosine triphosphate (p = 0.02) and intracellular pH (p = 0.04) in the initial 30 minutes of reperfusion was accelerated in the aprotinin-treated piglets. These piglets showed a greater in vivo cerebral and systemic endothelium-mediated vasodilation (acetylcholine response: cerebral p < 0.01, systemic p = 0.04) after reperfusion. The response to endothelium-independent vasodilation (nitroglycerin) was the same in both groups. Carotid blood flow tended to be greater at 20 minutes of reperfusion and less during 45 to 80 minutes after reperfusion in the aprotinin-treated animals. Brain water content postoperatively was 0.8077 in the aprotinin group and 0.8122 in control animals (p = 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A M3 muscarinic receptor coupled to inositol phosphate formation in the rat cochlea?

Various neuroactive substances, including excitatory and inhibitory amino acids, biogenic amines and neuropeptides, were tested for their ability to stimulate the inositol phosphate (IPs) cascade in the presence of lithium in the rat cochlea. Among them, only the muscarinic agonists (carbachol and oxotremorine M) were able to stimulate the IPs formation in 12-day-old rat cochleas. The carbachol-elicited IPs formation was inhibited by muscarinic antagonists with the following relative order of potency: atropine greater than 4-DAMP much greater than pirenzepine greater than methoctramine = AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the increase in IPs synthesis in the rat cochlea. However, an interaction with a m5 receptor subtype could not be completely excluded. The unusual link of only one receptor subtype with the phosphoinositide breakdown in the cochlea, as opposed to the usual existence of several receptors coupled to this transduction system in other organs such as the brain, suggest a unique role for muscarinic agonists in the cochlea.

Animals↗
PubMed2006

Cordycepin inhibits lipopolysaccharide-induced inflammation by the suppression of NF-kappaB through Akt and p38 inhibition in RAW 264.7 macrophage cells.

Cordyceps militaris, a caterpillar-grown traditional medicinal mushroom, produces an important bioactive compound, cordycepin (3'-deoxyadenosine). Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus and anti-infection activities. The molecular mechanisms of cordycepin on pharmacological and biochemical actions of macrophages in inflammation have not been clearly elucidated yet. In the present study, we tested the role of cordycepin on the anti-inflammation cascades in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. In LPS-activated macrophage, nitric oxide (NO) production was inhibited by butanol fraction of C. militaris and the major component of C. militaris butanol faction was identified as cordycepin by high performance liquid chromatography. To investigate the mechanism by which cordycepin inhibits NO production and inducible nitric oxide synthase (iNOS) expression, we examined the activation of Akt and MAP kinases in LPS-activated macrophage. Cordycepin markedly inhibited the phosphorylation of Akt and p38 in dose-dependent manners in LPS-activated macrophage. Moreover, cordycepin suppressed tumor necrosis factor (TNF-alpha) expression, IkappaB alpha phosphorylation, and translocation of nuclear factor-kappaB (NF-kappaB). The expressions of cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly decreased in RAW 264.7 cell by cordycepin. Taken together, these results suggest that cordycepin inhibits the production of NO production by down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB activation, Akt and p38 phosphorylation. Thus, cordycepin may provide a potential therapeutic approach for inflammation-associated disorders.

Animals↗
PubMed2006

Enhanced inflammatory response via activation of NF-kappaB in acute experimental diabetic neuropathy subjected to ischemia-reperfusion injury.

Reperfusion following ischemia increases ischemic fiber degeneration (IFD) in diabetic nerves compared to control normoglycemic nerves. The mechanism of this excessive susceptibility is unclear. Since reperfusion injury results in an inflammatory response, we tested the hypothesis that the diabetic state increases the inflammatory cascade. We used an animal model of unilateral ischemia-reperfusion (IR) injury to streptozotocin (STZ)-induced diabetic nerve to evaluate the density and localization of mediators of the inflammatory response using selective immunolabeling methods (for nuclear factor kappa B (NF-kappaB), intercellular adhesion molecule-1 (ICAM-1), cytokines and inflammatory cells). We studied a 1-month diabetic group and an age-matched control group (n=6 each). The right limb underwent 3 h ischemia at 35 degrees C and 7 days reperfusion. This was achieved by ligating the supplying arteries and collaterals to the right sciatic-tibial nerve for 3 h, followed by releasing the ties. Immunohistochemistry was performed on proximal sciatic and mid tibial nerves. NF-kappaB expression in diabetic sciatic endothelial cell and Schwann cell (SC) was significantly increased over that of controls subjected to identical IR injury. We observed a nearly 2-fold increase in density of NF-kappaB and ICAM-1 expression in microvessels of diabetic nerve compared with control nerve. Extensive infiltration of monocyte macrophages (1C7) was observed in the endoneurium of diabetic nerves, while only mild infiltration of granulocytes (HIS 48) occurred in the endoneurium of diabetic tibial nerves. This study provides evidence for an enhanced inflammatory response in diabetic nerves subjected to IR injury apparently via NF-kappaB activation.

Acute Disease↗
PubMed2001

Expression of JNK cascade scaffold protein JSAP1 in the mouse nervous system.

The mitogen-activated protein kinase (MAPK) cascades consist of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). The specificity of activation of MAPK cascades may be determined, in part, by scaffold proteins that organize multi-enzyme complexes. We have earlier reported a scaffold protein JSAP1 (also known as JIP3) in the JNK MAPK cascade. We also showed that, of the adult mouse tissues tested, JSAP1 mRNA was predominantly expressed in brain. Here we report the localization of JSAP1 protein in mouse embryos and adult brain by immunohistochemical analysis. In embryos (E11-16), JSAP1 immunoreactivity was mainly found in the central and peripheral nervous systems, where it was localized to the cell bodies and/or axons of developing neurons, but not neural precursor cells. In the adult brain, immunoreactive JSAP1 was localized mostly to cell bodies in almost all neurons. We also showed that the expression of JSAP1 transcripts and proteins gradually increased during the neural differentiation of mouse P19 embryonal carcinoma (EC) cells. Furthermore, we showed that overexpressed JSAP1 facilitated the efficient activation of JNK by MEKK1 in P19 cells. These results suggest that JSAP1 may function as a scaffold protein for the JNK signaling module in neuronal cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats.

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non-erythroid alpha II-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of alpha II-spectrin by calpain and caspase-3 results in accumulation of protease-specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of alpha II-spectrin and alpha II-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native alpha II-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of alpha II-spectrin and alpha II-SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.

Animals↗
PubMed2001

PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction.

BACKGROUND: Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. METHODS: To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. RESULTS: Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). CONCLUSIONS: These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

Animals↗
PubMed1997

A sweat gland-derived differentiation activity acts through known cytokine signaling pathways.

The sympathetic innervation of sweat glands undergoes a target-induced noradrenergic to cholinergic/peptidergic switch during development. Similar changes are induced in cultured sympathetic neurons by sweat gland cells or by one of the following cytokines: leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), or cardiotrophin-1 (CT-1). None of these is the sweat gland-derived differentiation activity. LIF, CNTF, and CT-1 act through the known receptors LIF receptor beta (LIFRbeta) and gp130 and well defined signaling pathways including receptor phosphorylation and STAT3 activation. Therefore, to determine whether the gland-derived differentiation activity was a member of the LIF/CNTF cytokine family, we tested whether it acted via these same receptors and signal cascades. Blockade of LIFRbeta inhibited the sweat gland differentiation activity in neuron/gland co-cultures, and extracts of gland-containing footpads stimulated tyrosine phosphorylation of LIFRbeta and gp130. An inhibitor (CGX) of molecules that bind the CNTFRalpha, which is required for CNTF signaling, did not affect the gland-derived differentiation activity. Soluble footpad extracts induced the same changes in NBFL neuroblastoma cells as LIF and CNTF, including increased vasoactive intestinal peptide mRNA, STAT3 dimerization, and DNA binding, and stimulation of transcription from the vasoactive intestinal peptide cytokine-responsive element. Thus, the sweat gland-derived differentiation activity uses the same signaling pathway as the neuropoietic cytokines, and is likely to be a family member.

Animals↗
PubMed1998

Hyperosmolality causes growth arrest of murine kidney cells. Induction of GADD45 and GADD153 by osmosensing via stress-activated protein kinase 2.

Murine kidney cells of the inner medullary collecting duct (mIMCD) were exposed to either isosmotic (300 mosmol/kg) or hyperosmotic medium (isosmotic medium + 150 mM NaCl) after seeding. We determined cell numbers, total nucleic acid, DNA, and RNA contents in both groups every day for a total period of 7 days. Based on all 4 parameters it was evident that growth of mIMCD3 cells is arrested for approximately 18 h following onset of hyperosmolality. However, none of the parameters measured indicated cell death because of hyperosmolality. Growth curves of hyperosmotic samples were shifted compared with isosmotic samples showing a gap of 18 h but had the same shape otherwise. We demonstrated that at 24 and 48 h after onset of hyperosmolality, but not in isosmotic controls, growth arrest and DNA damage-inducible (GADD) proteins GADD45 and GADD153 are strongly induced. This result is consistent with growth arrest observed in hyperosmotic medium. We tested if mitogen- and stress-activated protein kinase (SAPK) cascades are involved in osmosignaling that leads to GADD45 and GADD153 induction. Using phosphospecific antibodies we showed that extracellular signal-regulated kinases 1 and 2 (ERK), SAPK1 (JNK), and SAPK2 (p38) are hyperosmotically activated in mIMCD cells. Hyperosmotic GADD45 induction was significantly decreased by 37.5% following inhibition of the SAPK2 pathway, whereas it was significantly increased (65.2%) after inhibition of the ERK pathway. We observed similar, although less pronounced effects of SAPK2 and ERK inhibition on hyperosmotic GADD153 induction. In conclusion, we demonstrate that mIMCD cells arrest growth following hyperosmotic shock, that this causes strong induction of GADD45 and GADD153, that GADD induction is partially dependent on osmosignaling via SAPK2 and ERK, and that SAPK2 and ERK pathways have opposite effects on GADD expression.

Animals↗
PubMed2001

Deposition of nacystelyn from a dry powder inhaler in healthy volunteers and cystic fibrosis patients.

The aim of this study was to compare, using gamma scintigraphy, the lung deposition of a novel mucoactive agent, Nacystelyn (NAL), administered as a dry powder inhaler (DPI) in six healthy volunteers, six adult patients with cystic fibrosis (CF), and six children and adolescents patients with CF. The correlation between in vitro and in vivo results was also tested. It was first demonstrated that the method of labeling of NAL with 99mTc was reliable as tested by three in vitro methods (multistage liquid impinger, multistage cascade impactor, and 2-stage glass impinger). The deposition of unlabeled NAL, labeled NAL, and the radiolabel was similar in all stages of each device. Furthermore, the fine particle fraction (FPF) was the same on all apparatuses. The mean lung deposition obtained in volunteers was 27.5 +/- 13.5%. The results are approximately three times higher than the results obtained previously in healthy volunteers with NAL metered-dose inhalers (MDIs). As expected, the lung deposition observed in patients with CF was lower, e.g., 23.5 +/- 7.0% for adults and 16.5 +/- 5.9% for children and adolescents. A significant correlation was found between lung deposition and both the patient weight (p < 0.02) and height (p < 0.04). Surprisingly, the peripheral:central (P:C) ratio was similar for the three populations, indicating that the presence of mucus in moderately ill patients with CF does not modify the lung distribution of NAL. The FPF measured in vitro was similar to that obtained in volunteers but higher than that found in both patient populations. The DPI formulation of NAL developed will probably improve patient compliance and comfort in future clinical trials and postmarketing use of the drug.

Absorption↗
PubMed2004

Comparison of the TSI Model 3306 Impactor Inlet with the Andersen Cascade Impactor: solution metered dose inhalers.

The product performance of a series of solution Metered Dose Inhalers (MDIs) were evaluated using the TSI Model 3306 Impactor Inlet and the Andersen Cascade Impactor (ACI). The goal of the study was to test whether the fine particle and coarse particle depositions obtained using the Model 3306 were comparable to those results obtained by ACI testing. The analysis using the Model 3306 was performed as supplied by the manufacturer as well as with 20 cm and 40 cm vertical extensions that were inserted between the Model 3306 and the USP Inlet. Nine different solution formulations were evaluated. The drug concentrations ranged from 0.08 to 0.8% w/w and the ethanol cosolvent concentration varied between 5 and 20% w/w. In general, it was found that good correlations between the two instruments were obtained. However, for formulations containing 10-20% w/w ethanol it is shown that an extension fitted to the Model 3306 yielded an improved correlation to those obtained from the ACI.

Beclomethasone↗