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PubMed2005

Behavioral testing upregulates pCaMKII, BDNF, PSD-95 and egr-1 in hippocampus of FVB/N mice.

Several protein cascades are proposed to be involved in the formation of synaptic plasticity and have been linked to neuronal information processing and storage. Although modified expression of specific proteins following behavioral testing has been shown, no systematic approach for their concomitant determination has been reported. We therefore determined hippocampal expression of signaling proteins, transcription factors and synaptosomal-associated proteins representing key elements of neuronal plasticity in mice following behavioral training. Male FVB/N mice, 12 weeks of age, were used for behavioral testing. After completion of tests mice were sacrificed and hippocampi were dissected. Levels of total and autophosphorylated (T286) alphacalcium-calmodulin dependent kinase II (CaMKII, pCaMKII), total and phosphorylated mitogen-activated protein kinase (MAPK, pMAPK), total and phosphorylated calcium-responsive element binding (creb, pcreb), early-growth response protein 1 (egr-1), brain derived neurotrophic factor (BDNF), tyrosine kinase receptor B (trk B), drebrin and postsynaptic density-95 (PSD-95) were quantified in hippocampi of behavior trained animals (n=7) and naïve caged controls (n=7). Expression of pCaMKII, BDNF, PSD-95 and egr-1 was significantly increased in the behavior-trained group. Expression of total CaMKII, total and pMAPK, total and pcreb, trk B and drebrin was comparable between groups. Detection of significantly increased pCaMKII, BDNF, PSD-95 and egr-1 induced by behavioral training at the protein level per se is intriguing and supports the proposed importance of these molecules for neuronal information storage.

Animals↗
PubMedDate not supplied

CASCADE: a European collaborative study on vascular determinants of brain lesions. Study design and objectives.

Dementia is a highly prevalent disease that may have a cardiovascular component. White matter lesions and brain atrophy (brain abnormalities) are prevalent in dementia cases and might form part of the anatomical basis for the disease. We designed a multi-centre study, CASCADE (Cardiovascular Determinants of Dementia), to examine long-term (10-20 years) and short-term (5 years) cardiovascular risk factors for, and the cognitive consequence of, brain abnormalities. White matter lesions and atrophy are measured with magnetic resonance imaging. Cognitive function is measured with nine tests of memory and executive function. The studies included in CASCADE were ongoing and geographically spread throughout Europe to capture the cardiovascular risk gradient. In each study, a random sample of at least 100 subjects aged 65-75 years was selected who participated in the previous research examinations conducted by the respective centres. The objectives and design of the CASCADE project are described.

Aged↗
PubMed1998

Buprenorphine hydrochloride induces apoptosis in NG108-15 nerve cells.

A morphine alkaloid derivative, buprenorphine hydrochloride, induces apoptosis in NG108-15 cells. Apoptosis was detected mainly by apoptosis-specific DNA fragmentation and morphological changes. This apoptosis was dose-dependent and the time-course experiment indicated that DNA fragmentation occurred within 4 h after administration of buprenorphine hydrochloride. Specific inhibitors of the previously characterized apoptotic signal cascade as well as antagonists for opioid receptors were tested. Zn2+, herbimycin A, caspase inhibitors YVAD (Ac-Tyr-Val-Ala-Asp-CHO) and DEVD (Ac-Asp-Glu-Val-Asp-CHO), naloxone and naltrindole had no effect on apoptosis-specific DNA fragmentation. The serine protease inhibitor TPCK (N-tosyl-L-phenylalanyl chloromethyl ketone) specifically inhibited apoptosis-specific DNA fragmentation induced by buprenorphine hydrochloride; however, cell viability measurements revealed that cell death still occurred in NG108-15 cells. Thus TPCK pretreatment before buprenorphine hydrochloride administration induced apoptosis-independent cell death, presumably necrosis, in NG108-15 cells. This suggests that an unidentified serine protease, presumably functioning in the buprenorphine hydrochloride-specific death-signal cascade, could be pivotal for the rapid apoptosis observed in NG108-15 cells upon treatment with buprenorphine hydrochloride.

Analgesics, Opioid↗
PubMed1994

Nuclear receptor steroidogenic factor 1 regulates the müllerian inhibiting substance gene: a link to the sex determination cascade.

Normal male sex differentiation requires that Sertoli cells in the embryonic testes produce müllerian inhibiting substance (MIS), a TGF beta-like hormone that causes müllerian duct regression. In primary Sertoli cells, the orphan nuclear receptor, steroidogenic factor 1 (SF-1), regulates the MIS gene by binding to a conserved upstream regulatory element. In heterologous (HeLa) cells, MIS gene activation by SF-1 requires removal of the SF-1 ligand-binding domain, implicating a Sertoli cell-specific ligand or cofactor. Finally, the sexually dimorphic expression of SF-1 during development coincides with MIS expression and müllerian duct regression. We propose that SF-1 regulates MIS in vivo and participates directly in the process of mammalian sex determination.

Animals↗
PubMed2001

LH/chorionic gonadotropin signaling pathway involves protein tyrosine phosphatase activity downstream of protein kinase A activation: evidence of an obligatory step in steroid production by Leydig cells.

Our recent reports indicate that protein tyrosine phosphorylation is an obligatory component of the mechanism of action of ACTH in its stimulatory action of corticosteroid production in adrenal zona fasciculata (ZF). The role of protein tyrosine phosphatase (PTP) activity in the regulation of steroidogenesis by LH/chorionic gonadotropin (CG) was tested using cell-permeable PTP inhibitors. Thus, PTP inhibition blocks LH- and 8-bromo-cAMP-stimulated testosterone production by Leydig cells without affecting 22(R)OH-cholesterol-supported steroidogenesis, similar results to those obtained in the adrenal ZF/ACTH system, leading us to propose that PTP action is an obligatory and common step in the cascade triggered by both hormones. Then, we continued the study testing whether LH modulates PTP activity in MA-10 cells, a Leydig cell line. In this regard, we observed by an in-gel PTP assay two PTPs of 110 and 50 kDa that are activated by hormone and 8-bromo-cAMP activation of the cells. Moreover, there is a transient increase by the second messenger in total PTP activity that correlates with the higher activity displayed by the 110 and 50 kDa proteins in the in-gel assay. In accordance with these results, analysis of tyrosine phosphorylated proteins showed the LH-induced dephosphorylation of proteins of 120, 68 and 50 kDa. The results of this study indicate that PTPs play an important role in the regulation of Leydig cell functions and that there exists a cross talk between serine/threonine phosphorylation and tyrosine dephosphorylation mediated by hormone-activated cAMP-dependent protein kinase and PTPs. These results are the first evidence of PTP having a role in LH/CG-stimulated steroidogenesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Cardiac-specific overexpression of diacylglycerol kinase zeta prevents Gq protein-coupled receptor agonist-induced cardiac hypertrophy in transgenic mice.

BACKGROUND: Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqalpha protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. METHODS AND RESULTS: To test this hypothesis, we generated transgenic (DGKzeta-TG) mice with cardiac-specific overexpression of DGKzeta. There were no differences in heart size and heart weight between DGKzeta-TG and wild-type littermate mice. The left ventricular function was normal in DGKzeta-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKzeta-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKzeta-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKzeta-TG mouse hearts, suggesting that DGKzeta regulated PKC activity by controlling cellular diacylglycerol levels. CONCLUSIONS: These results demonstrated the first evidence that DGKzeta negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.

Angiotensin II↗
PubMed2000

A novel express bioassay for detecting toxic substances in water by recording rhodopsin-mediated photoelectric responses in Chlamydomonas cell suspensions.

The influence of Cu2+, Zn2+, Cd2+, Pb2+ and formaldehyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was investigated using three modifications of a recently developed population method for electrical recording (in nonoriented, phototactically preoriented (PO) and gravitactically preoriented cell suspensions). The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the responses. Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simultaneously with an inhibition of their photoelectric cascade, photo-orientation and motility. The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive phototaxis in the presence of the toxic substances. Of the two major components of the photoelectric cascade, the regenerative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the photoreceptor current. The results obtained show that measurement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bioassay for testing environmental pollutants in water samples.

Animals↗
PubMed1998

Angiotensin II and angiotensin-converting enzyme as candidate compounds modulating the effects of testis ecdysiotropin in testes of the gypsy moth, Lymantria dispar1.

Lymantria dispar testes synthesize immunodetectable ecdysteroid in vitro in response to the brain peptide, testis ecdysiotropin (TE), acting primarily via a cascade involving Gi protein, diacyl glycerol, and phosphokinase C. However, a component of TE activation also involves the opposite cascade, Gs protein, cAMP, and phosphokinase A. Excess cAMP inhibits the action of TE, acting as a feedback modulator. Here, we show that bovine angiotensin II (AII) and bovine angiotensin converting enzyme (ACE) act like cAMP, inducing synthesis of immunodetectable ecdysteroid by pupal testes in vitro, but are antagonistic to coincubated TE. In addition, an insect ACE antibody clearly stains the spermatogenic cells through all stages of development, as well as testis sheath tissue where ecdysteroid is synthesized. AII induces synthesis of cAMP by pupal testes in vitro. Therefore, insect homologs of mammalian AII and ACE are good candidates for the peptides responsible for the cAMP cascade and as modulators of TE action in lepidopteran testes. Saralasin, an analog of AII that blocks angiotensin receptors in mammals, behaved like AII in inducing ecdysteroid secretion with ecdysteroidogenic effects additive to either angiotensin or ACE. Therefore, the receptors for the insect form of angiotensin on lepidopteran testis cells are probably different from those in mammals. Saralasin also inhibited ecdysteroid synthesis when combined with TE, as did AII.

Angiotensin II↗
PubMed1999

Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition.

Fully grown oocytes of most laboratory mice progress without interruption from the germinal vesicle (GV) stage to metaphase II, where meiosis is arrested until fertilization. In contrast, many oocytes of strain LT mice arrest precociously at metaphase I and often undergo subsequent spontaneous parthenogenetic activation. Cytostatic factor (CSF), which prevents the degradation of cyclin B and maintains high maturation-promoting factor (MPF) activity, is required for maintenance of metaphase I-arrest in LT oocytes, similar to its requirement for maintaining metaphase II-arrest in normal oocytes. However, CSF does not instigate metaphase I-arrest since a temporary metaphase I-arrest occurs in MOS-null LT oocytes. This paper addresses the mechanism(s) that may instigate metaphase I-arrest and tests the hypothesis that there may be one or more defects in LT oocytes that delay their acquisition of competence to trigger the cascade of processes that normally drive entry into and progression through anaphase I. To test this hypothesis, MPF activity was artificially abrogated by treating oocytes with a general protein kinase inhibitor, 6-DMAP, at various times during the progression of meiosis I. This allowed a comparison of the time at which LT and normal oocytes become competent to undergo the metaphase I/anaphase transition even if oocytes were arrested at metaphase I when 6-DMAP-treatment was begun. There were no differences between LT and control oocytes in the kinetics of MPF suppression by 6-DMAP. However, it was found that LT oocytes do not acquire competence to undergo the metaphase I/anaphase transition in response to 6-DMAP until 50-60 min after normal oocytes. A similar delay was observed in strain CX8-4 oocytes, which also have a high incidence of metaphase I-arrest, but not in strain CX8-11 oocytes, which exhibit a low incidence of metaphase I-arrest. MOS-null LT oocytes also exhibit a delay in acquisition of competence to undergo the metaphase I/anaphase transition. Thus, a delay in competence to undergo the metaphase I/anaphase transition in response to 6-DMAP-treatment correlates with metaphase I-arrest. It is therefore hypothesized that the observed delay in acquisition of competence to enter anaphase I may instigate the sustained metaphase I-arrest in LT oocytes by allowing CSF activity to rise to a level that prevents cyclin B degradation and maintains high MPF activity before anaphase can be initiated by normal triggering mechanisms.

Adenine↗
PubMed2006

Role of Toll-like receptor 4 for the pathogenesis of acute lung injury in Gram-negative sepsis.

BACKGROUND AND OBJECTIVE: Proinflammatory cytokines as well as nitric oxide (NO) play a major role in mediating the response to lipopolysaccharide (LPS). The present study tested the hypothesis that LPS induces proinflammatory cytokines in the lung via the Toll-like receptor 4 (TLR4)/CD14 signalling cascade. METHODS: Control mice and TLR4-deficient (TLR4-D) mice were used to test TLR4-mediated effects of LPS. Both strains received either Escherichia coli LPS (20 mg kg-1 intraperitoneal) or saline and their lungs were collected at different time points. Pulmonary nuclear factor kappaB (NFkappaB) activation was investigated with electromobility shift assay. mRNA expression of inflammatory mediators and their corresponding receptors were detected with Ribonuclease Protection Assay. Protein expression was detected by ELISA and western blotting. Inducible NO synthase (iNOS) expression was monitored by RT-PCR and iNOS activity by conversion of l-arginine to citrulline. Immune cells were sampled by bronchoalveolar lavage (BAL) and classified. RESULTS: LPS application induced CD14-, but not TLR4 protein expression in control mice. Activation of pulmonary NFkappaB was observed within 60 min in control, but not in TLR4-D mice. Six hours of LPS administration induced a significant increase in pulmonary tumour necrosis factor alpha-, interleukin-1beta- and interleukin-6 mRNA and protein expression in control mice compared to TLR4-D mice. Furthermore, LPS induced a significantly higher increase of the iNOS expression and catalytic activity in control mice than in TLR4-D mice. BAL revealed an increase in total cell count in all LPS treated mice. CONCLUSION: Our findings suggest that TLR4 plays a key role for regulating the expression of relevant cytokines within the lung during endotoxic shock.

Animals↗
PubMed1989

Preferential support of Ca2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade.

Studies of intact smooth muscle have suggested that its anomalous aerobic lactate production may reflect an intracellular compartmentation of glycolytic enzyme cascades designed to support specific exergonic processes. In particular, we have postulated a membrane-associated glycolytic cascade that preferentially supports the ATP requirements of membrane functions. We tested this hypothesis by using a smooth muscle plasma membrane fraction (PMV) purified for calcium pump activity. We show that glycolytic enzymes are endogenous in PMV and can produce NADH, ATP, and lactate from fructose 1,6-diphosphate in the presence of glycolytic cofactors. This glycolytic cascade can fuel the calcium pump despite the presence of an ATP trap that eliminated calcium uptake fueled by exogenously added ATP. This plasma membrane glycolytic cascade is coupled to calcium pump function in a tissue with both oxidative and glycolytic metabolism. Thus coupling of metabolic cascades with the specific processes they subserve may be a more general feature of cellular organization than was previously thought.

Adenosine Triphosphate↗
PubMed1992

Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

Cyclic AMP (cAMP) is the major intracellular second messenger of thyrotropin (TSH) action on thyroid cells. It stimulates growth as well as the function and differentiation of cultured thyrocytes. The adenosine A2 receptor, which activates adenylyl cyclase via coupling to the stimulating G protein (Gs), has been shown to promote constitutive activation of the cAMP cascade when transfected into various cell types. In order to test whether the A2 receptor was able to function similarly in vivo and to investigate the possible consequences of permanent adenylyl cyclase activation in thyroid cells, lines of transgenic mice were generated expressing the canine A2 adenosine receptor under control of the bovine thyroglobulin gene promoter. Thyroid-specific expression of the A2 adenosine receptor transgene promoted gland hyperplasia and severe hyperthyroidism causing premature death of the animals. The resulting goitre represents a model of hyperfunctioning adenomas: it demonstrates that constitutive activation of the cAMP cascade in such differentiated epithelial cells is sufficient to stimulate autonomous and uncontrolled function and growth.

Adenosine↗
PubMed2000

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Alternative Splicing↗
PubMed1992

Importance of bicarbonate ion for intracellular pH regulation in antigen- and ionomycin-stimulated RBL-2H3 mast cells.

In RBL-2H3 rat basophilic leukemia cells, Ca2+ influx and secretion are activated by antigens that crosslink IgE-receptor complexes and by the Ca2+ ionophore, ionomycin. Here we report that antigen-stimulated Ca2+ influx and secretion are impaired and ionomycin-induced responses are strongly inhibited following the removal of HCO3- from the medium. These results raised the possibility that HCO3(-)-dependent pH regulation mechanisms play a role in the cascade of events leading to mast cell activation. To test this hypothesis, intracellular pH (pHi) was measured by ratio imaging microscopy in individual RBL-2H3 cells labeled with 2',7'-bis-(2-carboxyethyl)-5-(6) carboxyfluorescein (BCECF). In unstimulated cells, it was found that basal pHi in the presence of HCO3- is 7.26, significantly greater than pHi in its absence, 7.09 (P less than 10(-6]. These results, as well as evidence that pHi increases rapidly when HCO3- is added to cells initially incubated in HCO3(-)-free medium, indicate that unstimulated cells use a HCO3(-)-dependent mechanism to maintain cytoplasmic pH. Further analyses comparing unstimulated with stimulated cells showed that antigen causes a small transient acidification in medium containing HCO3- and a larger sustained acidification in HCO3(-)-depleted medium. Ionomycin is a more potent acidifying agent, stimulating a sustained acidification in complete medium and causing further acidification in HCO3(-)-free medium. These results support the hypothesis that the inhibition of antigen- and ionomycin-induced 45Ca2+ influx and secretion in cells incubated in HCO3(-)-free medium is at least partially due to the inactivation of HCO3(-)-dependent mechanisms required to maintain pH in unstimulated cells and to permit pH recovery from stimulus-induced acidification.

Animals↗
PubMed2003

Role and expression of thrombin receptor PAR-1 in muscle cells and neuromuscular junctions during the synapse elimination period in the neonatal rat.

A role for thrombin and its receptor (ThR) during mammalian skeletal muscle cell differentiation and neuromuscular junction (NMJ) formation has been suggested. Previously, we found that the synapse elimination process in the neonatal rat muscle was accelerated by thrombin and blocked by hirudin, its specific inhibitor (Lanuza et al. [2001] J. Neurosci. Res. 63:330-340). To test whether this process resulted from a signal transduction cascade initiated by activation of ThR, in particular PAR-1, we applied to the levator auris longus (LAL) muscle of newborn rats two synthetic peptides (SFLL and FSLL). SFLL is a potent specific agonist for activation of PAR-1, whereas FSLL is an inactive peptide. We have demonstrated that the activation of PAR-1 by SFLL produced acceleration of the presynaptic loss of connections and the postsynaptic maturation of NMJs. Moreover, Western blot analysis showed that PAR-1 was present in the skeletal muscle, and by immunohistochemistry we detected PAR-1 in muscle fibers concentrated in the synaptic area but also in satellite cells. Several lines of evidence suggested that PAR-1 is localized in the postsynaptic membrane: PAR-1 immunofluorescence was concentrated at denervated synaptic sites and was present in the myotube membrane in vitro in the absence of neurons and in dissociated single muscle fibers from which nerve terminals and Schwann cells had been removed. Taken together, these results indicate that thrombin mediates certain stages of activity-dependent synapse elimination in the skeletal muscle and does so through its action on the thrombin receptor PAR-1 localized, at least in part, on the postsynaptic membrane.

Animals↗
PubMed2006

Medial collateral ligament healing in macrophage metalloelastase (MMP-12)-deficient mice.

Medial collateral ligament (MCL) injuries heal by a wound repair scar response controlled by a complex cellular and cytokine environment. Many enzymes participate in wound repair, particularly the matrix metalloproteinases. We hypothesize macrophage metalloelastase (MME/MMP-12) deficiency results in impaired healing of MCL injury. One hundred fifty MME-deficient and 150 WT (MME+/+) mice underwent knee MCL transection with the opposite knee as a sham operated control. Mice were sacrificed at 3, 7, 28, 42, and 56 days. At each of the five time points, 15 mice were utilized for biological and 15 were utilized for biomechanical testing. Outcome measures were the presence of macrophages to represent the inflammatory phase of wound healing, collagen synthesis to assay for matrix repair, and biomechanical testing for repair strength. Immunohistochemistry demonstrated significantly fewer macrophages in cut MCLs from MME-deficient mice versus wild-type (WT) mice at 3, 7, 28, and 42 days (all p<or=0.04). In situ hybridization to Col1a1 mRNA in the MME-deficient cut MCLs at 7, 28, and 42 day time points showed a decreased level of type I pro-collagen mRNA compared to the WT cut MCLs (p<0.05). Biomechanical testing revealed cut ligaments from MME-deficient mice had significantly lower ultimate force and stiffness compared to cut ligaments from WT mice (p<0.001), with maximal differences of 40% at 7 days for ultimate force and 28 days for stiffness (p<0.05 by Tukey post hoc test). We conclude MME is important in the multifactorial cascade of knee MCL injury healing, showing significant differences in both the early inflammatory and in the matrix tissue synthesis phases.

Animals↗
PubMed2000

Differential patterns of ERK and STAT3 phosphorylation after sciatic nerve transection in the rat.

Peripheral nerve injury induces a specific pattern of expression of growth factors and cytokines, which regulate injury responses and regeneration. Distinct classes of growth factors and cytokines signal through specific intracellular phosphorylation cascades. For example, the ERK phosphorylation cascade mediates signaling through transmembrane tyrosine kinase receptors and the JAK/STAT cascade mediates signaling through the GP130 receptor complex. We tested whether specific phosphorylation patterns of ERK and STAT3 result from nerve injury and whether such phosphorylation correlates with the expression of specific growth factors and cytokines. At sites adjacent to a nerve transection, we observed that ERK phosphorylation peaked early, persisted throughout 16 days, and was equally intense at proximal and distal sites. In contrast, STAT3 phosphorylation peaked later than ERK but did not persist as long and was stronger in the proximal than in the distal segment adjacent to the injury. In addition, in distal segments further away from the injury site, ERK became phosphorylated with a delayed time course, while STAT3 remained unphosphorylated. These patterns of phosphorylation correlated well with the expression of neurotrophin and interleukin-6 mRNAs in the distal stump. In addition, we found that the pattern of SAPK phosphorylation is similar to the pattern observed for STAT3, while the pattern of macrophage infiltration into the transected nerve was distinct from all the phosphorylation patterns observed. Together, these observations suggest that ERK activation is important in the establishment of a regeneration-promoting extracellular environment in the far distal stump of transected nerves and that STAT3 activation is important in the control of cellular responses close to the site of injury.

Animals↗
PubMed1996

Regulation of Na-K-ATPase activity in the proximal tubule: role of the protein kinase C pathway and of eicosanoids.

To evaluate further the signal transduction mechanisms involved in the short-term modulation of Na-K-ATPase activity in the mammalian kidney, we examined the role of phospholipase C-protein kinase C (PLC-PKC) pathway and of various eicosanoids in this process, using microdissected rat proximal convoluted tubules. Dopamine (DA) and parathyroid hormone (either synthetic PTH1-34 or PTH3-34) inhibited Na-K-ATPase activity in dose-dependent manner; this effect was reproduced by PKC530-558 fragment and blocked by the specific PKC inhibitor calphostin C, as well as by the PLC inhibitors neomycin and U-73122. Pump inhibition by DA, PTH, or arachidonic acid, and by PKC activators phorbol dibutyrate (PDBu) or dioctanoyl glycerol (DiC8) was abolished by ethoxyresorufin, an inhibitor of the cytochrome P450-dependent monooxygenase pathway, but was unaffected by indomethacin or nordihydroguaiaretic acid, inhibitors of the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, respectively. Furthermore, each of the three monooxygenase products tested (20-HETE, 12(R)-HETE, or 11,12-DHT) caused a dose-dependent inhibition of the pump. The effect of DA, PTH, PDBu or DiC8, as well as that of 20-HETE was not altered when sodium entry was blocked with the amiloride analog ethylisopropyl amiloride or increased with nystatin. We conclude that short-term regulation of proximal tubule Na-K-ATPase activity by dopamine and parathyroid hormone occurs via the PLC-PKC signal transduction pathway and is mediated by cytochrome P450-dependent monooxygenase products of arachidonic acid metabolism, which may interact with the pump rather than alter sodium access to it.

Amiloride↗