Biomedical subjects

M Kress

Publications and source records attributed to M Kress.

At least 19 recordsLinked to original sources

PubMed2005

Fast modulation of heat-activated ionic current by proinflammatory interleukin 6 in rat sensory neurons.

The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.

Acetophenones↗

PubMed2005

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules.

The cytoplasmic polyadenylation element-binding protein (CPEB) has been characterized in Xenopus laevis as a translational regulator. During the early development, it behaves first as an inhibitor and later as an activator of translation. In mammals, its closest homologue is CPEB1 for which two isoforms, short and long, have been described. Here we describe an additional isoform with a different RNA recognition motif, which is differentially expressed in the brain and ovary. We show that all CPEB1 isoforms are found associated with two previously described cytoplasmic structures, stress granules and dcp1 bodies. This association requires the RNA binding ability of the protein, whereas the Aurora A phosphorylation site is dispensable. Interestingly, the rck/p54 DEAD box protein, which is known as a CPEB partner in Xenopus and clam, and as a component of dcp1 bodies in mammals, is also present in stress granules. Both stress granules and dcp1 bodies are involved in mRNA storage and/or degradation, although so far no link has been made between the two, in terms of neither morphology nor protein content. Here we show that transient CPEB1 expression induces the assembly of stress granules, which in turn recruit dcp1 bodies. This dynamic connection between the two structures sheds new light on the compartmentalization of mRNA metabolism in the cytoplasm.

Alternative Splicing↗

PubMed2004

Differences in calcium signalling in rat peripheral sensory neurons.

Calcium influx and the resulting increase in intracellular calcium concentration [Ca2+]i can induce enhanced sensitivity to temperature increases in nociceptive neurons. Using the patch-clamp technique and simultaneous calcium microfluorimetry we show that experimental elevation of [Ca2+]i using the calcium ionophore ionomycin resulted in a significant potentiation of heat-activated currents. This was not the case when rises in [Ca2+]i were elicited by depolarization of the cell membrane by current injection via the patch pipette. Our data provide first, however, indirect evidence that in sensory neurons calcium ions may be guided into different intracellular microdomains depending on the type of ion channel or pore through which they enter the cell. We conclude that the compartmentalization of sensory neurons for calcium ions may be decisive on further signalling cascades accounting, for example, for neuronal plasticity.

Animals↗

PubMed2003

The inflammatory mediators serotonin, prostaglandin E2 and bradykinin evoke calcium influx in rat sensory neurons.

The inflammatory mediators bradykinin, prostaglandin E(2) and serotonin interact to excite and sensitize nociceptive neurons. All three mediators are coupled to signaling pathways that potentially induce rises in intracellular calcium concentration in other models. The aim of this study was therefore to investigate if the three mediators cause calcium rises in isolated rat sensory neurons that may explain their sensitizing action. Neurons exposed to serotonin, bradykinin, and prostaglandin E(2) exhibited reversible increases in intracellular calcium concentration, which were absent in calcium-free solution. The calcium increase induced by serotonin was preserved in the presence of extracellular cadmium suggesting calcium influx potentially through the serotonin receptor ion channel 5-HT(3). The bradykinin-induced calcium response was slower, showed pronounced tachyphylaxis and was absent in the presence of extracellular cadmium ions. Similar results were obtained for prostaglandin E(2) although the calcium rises were fast and not prone to tachyphylaxis. This suggests that prostaglandin E(2) as well as bradykinin via activation of G protein-coupled receptors seem to couple to calcium-permeant ion channels possibly the heat-transducing vanilloid receptor type 1 or related ion channels. The three mediators, however, did not cooperate to induce supra-additive calcium responses when applied simultaneously. In summary, our results suggest that the inflammatory mediators serotonin, prostaglandin E(2) and bradykinin induce calcium influx in sensory neurons. However, they do not utilize a calcium-dependent cooperative mechanism to facilitate proton-induced currents.

Animals↗

PubMed2003

In vitro and in vivo interactions between the hepatitis B virus protein P22 and the cellular protein gC1qR.

gC1qR, a mitochondrial matrix protein, was identified as the main cellular partner of the hepatitis B virus P22 protein. We demonstrated by immunofluorescence studies that some P22 molecules were colocalized with the endogenous gC1qR in both the cytoplasm and the nucleus but never in the mitochondria. We also showed that the last 34 amino acids of P22 were involved in the association with gC1qR.

Amino Acid Sequence↗

PubMed2003

Fast Ca2+-induced potentiation of heat-activated ionic currents requires cAMP/PKA signaling and functional AKAP anchoring.

Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.

Adenylyl Cyclases↗

PubMed2002

The effects of excessive heat on heat-activated membrane currents in cultured dorsal root ganglia neurons from neonatal rat.

The effects of high temperature (53-61 degrees C) on membrane currents (I(heat)) or depolarization (V(heat)) induced by noxious heat were studied in cultured dorsal root ganglia neurons from neonatal rats using the whole cell patch clamp technique. I(heat) or V(heat) produced by 3 s ramps of increasing temperature between 43 and 50 degrees C exhibited a fast slope (Q10>10) that was similar both during rising and falling temperature (n=85). Temperatures exceeding 52 degrees C resulted in slowdown in the recovery of I(heat), and the threshold for inducing I(heat) was shifted to lower temperatures in successive trials. These high temperatures (54-60 degrees C) caused a linear and incomplete recovery of I(heat) (Q10 decreased to <5; 4.5 +/- 0.4; n=17) and in successive trials the threshold of I(heat) decreased to temperatures close to that in the bath. The neurons, however, remained sensitive to capsaicin and to decreased extracellular pH. It is suggested that exposure of nociceptive neurons to excessive noxious heat results in an irreversible decrease of the energy barrier between the resting and activated state of the protein structures responsible for generation of I(heat). This may explain the sensitization of nociceptors after heat injury.

Animals↗

PubMed2002

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Calcium Phosphates↗

PubMed2001

N- and L- but not P/Q-type calcium channels contribute to neuropeptide release from rat skin in vitro.

Here we directly demonstrate the liberation of CGRP from rat skin in vitro induced by high extracellular concentrations of KCl. The EC50 was 52 mM KCl and saturation was reached from 80 mM KCl. The release was entirely dependent on the presence of extracellular calcium ions. It was reduced by nonsubtype selective inhibition of voltage-operated calcium channels (VOCCs). Application of selective antagonists suggest expression of L-type and N-type but not P/Q-type VOCCs in cutaneous nociceptive terminals. These may be activated by any suprathreshold depolarizing stimuli to induce neurogenic inflammation. The expression pattern greatly differs from central nociceptive terminals where, in addition, P/Q-type VOCC have been found.

Animals↗

PubMed2001

The zinc finger transcription factor, MOK2, negatively modulates expression of the interphotoreceptor retinoid-binding protein gene, IRBP.

The human and murine MOK2 orthologue genes encode Krüppel/TFIIIA-related zinc finger proteins, which are factors able to recognize both DNA and RNA through their zinc finger motifs. MOK2 proteins have been shown to bind to the same 18-base pair (bp)-specific sequence in duplex DNA. This MOK2-binding site was found within introns 7 and 2 of human PAX3 and interphotoreceptor retinoid-binding protein (IRBP) genes, respectively. As these two genes are expressed in the brain as MOK2, we have suggested that PAX3 and IRBP genes are two potentially important target genes for the MOK2 protein. In this study, we focused our attention on IRBP as a potential MOK2 target gene. Sequence comparison and binding studies of the 18-bp MOK2-binding sites present in intron 2 of human, bovine, and mouse IRBP genes show that the 3'-half sequence is the essential core element for MOK2 binding. Very interestingly, 8-bp of this core sequence are found in a reverse orientation, in the IRBP promoter. We demonstrate that MOK2 can bind to the 8-bp sequence present in the IRBP promoter and repress its transcription when transiently overexpressed in retinoblastoma Weri-RB1 cells. In the IRBP promoter, it appears that the TAAAGGCT MOK2-binding site overlaps with the photoreceptor-specific CRX-binding element. We suggest that MOK2 represses transcription by competing with the cone-rod homeobox protein (CRX) for DNA binding, thereby decreasing transcriptional activation by CRX. Furthermore, we show that Mok2 expression in the developing mouse and in the adult retina seems to be concordant with IRBP expression.

Animals↗

PubMed2001

Pro- and anti-inflammatory actions of ricinoleic acid: similarities and differences with capsaicin.

We have investigated the pro- and anti-inflammatory effects of ricinoleic acid (RA), the main active principle of castor oil, in an experimental model of blepharitis induced by intradermal injection of carrageenan in the guinea-pig eyelid and its possible capsaicin-like mode of action on acutely dissociated rat dorsal root ganglia (DRG) neurons in vitro. Topical treatment with RA (10-100 mg/guinea-pig) or capsaicin (1-10 mg/guinea-pig) caused eyelid reddening and oedema. At lower doses (0.3-3 mg/guinea-pig and 0.009-0.09 mg/guinea-pig for RA and capsaicin, respectively) both drugs significantly potentiated the eyelid oedema induced by carrageenan. The tachykinin NK1 receptor antagonist FK 888 (0.59 mg/kg s.c.) abolished the potentiation of carrageenan-induced eyelid oedema induced by either RA or capsaicin. The neutral endopeptidase inhibitor, thiorphan (1.3 mg/kg i.v.) significantly enhanced the potentiation of carrageenan-induced eyelid oedema produced by RA. This potentiating effect was abolished by FK 888. Repeated (8 days) topical application of RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-pig) inhibited the carrageenan-induced eyelid oedema. This anti-inflammatory effect was accompanied by a reduction (75%-80% of SP and 46%-51% of NKA) in tachykinin content of the eyelids, as determined by radioimmunoassay. In dissociated rat DRG neurons, RA (0.1 mM for 5 min) significantly inhibited the inward currents induced by application of capsaicin (1 microM) and/or low pH (5.8), without inducing any currents by itself or changing voltage-dependent currents. Moreover, after 24-h incubation, RA (0.1 mM) significantly decreased the capsaicin (1 microM)-induced calcitonin gene-related peptide (CGRP) release from rat DRG neurons, whereas acute drug superfusion did not evoke CGRP release by itself. Summarizing, RA possesses capsaicin-like dual pro-inflammatory and anti-inflammatory properties which are observed upon acute and repeated application, respectively. However, unlike capsaicin, RA does not induce inward current in DRG neurons and it is devoid of algesic properties in vivo.

Animals↗

PubMed2001

Molecular physiology of proton transduction in nociceptors.

Recent cloning efforts have identified families of ion channels that may be involved in signaling noxious proton accumulation in tissue. Some conduct potassium ions outward and are closed by excess protons, others are opened under this condition carrying cations inward and their putative function is in their name ('acid sensing'), and again another channel is truly polymodal, the capsaicin receptor, sensing acid and heat. Further heat-activated channels, not yet cloned, may not be gated by protons but sensitized so strongly that they open at the command of body temperature. In either case, the result may be pain from tissue acidosis.

Animals↗

PubMed2001

Infection by human varicella-zoster virus confers norepinephrine sensitivity to sensory neurons from rat dorsal root ganglia.

Varicella-zoster virus (VZV) is a widespread human herpes virus causing chicken pox on primary infection and persisting in sensory neurons. Reactivation causes shingles, which are characterized by severe pain and often lead to postherpetic neuralgia. To elucidate the mechanisms of VZV-associated hyperalgesia, we elaborated an in vitro model for the VZV infection of sensory neurons from rat dorsal root ganglia. Between 35 and 50% of the neurons showed strong expression of the immediate-early virus antigens IE62 and IE63 and the late glycoprotein gE. When the intracellular calcium concentration was monitored microfluorometrically for individual cells after infection, the sensitivity to GABA or capsaicin was similar in controls and in VZV-infected neurons. However, the baseline calcium concentration was increased. Neurons became de novo sensitive to adrenergic stimulation after VZV infection. Norepinephrine-responsive neurons were more frequent and calcium responses to norepinephrine were significantly higher after infection with wild-type isolates than with the attenuated vaccine strain OKA. The adrenergic agonists phenylephrine and isoproterenol had similar efficacy. We suggest that the infection with wild-type VZV isolates confers norepinephrine sensitivity to sensory neurons by using alpha(1)- and/or beta(1)-adrenergic receptors providing a model for the pathophysiology of the severe pain associated with the acute reactivation of VZV.

Animals↗

PubMed2001

The determination of free and protein-bound haemoglobin in plasma using a combination of HPLC and absorption spectrometry.

The aim of the study was to develop a method for the determination of haemoglobin in plasma suitable for use to set target values for external quality assessment schemes for this analyte using commercially available test kits and equipment. In the early phase of the method development it became clear that the use of a single method, namely HPLC, would not be possible. However, by combining HPLC and absorption spectrophotometry, both qualitative and quantitative rapid determinations of protein-bound and free haemoglobin were able to be performed on equipment present in most routine clinical chemistry laboratories. The separation of protein-bound and free haemoglobin could be carried out using commercial HPLC equipment for the determination of haemoglobin A1c (HbA1c) without modification of the conditions used. Instead of haemolysed blood, the same volume of plasma (10 microl) was injected. The eluate was not discarded, but collected in 1-minute fractions so that the void volume (protein-bound Hb) and the haemoglobin peaks (free Hb) were available for the colorimetric determination of haemoglobin using the pseudoperoxidase activity of the haem moiety on hydrogen peroxide and a chromogen (3,3',5,5'-tetramethylbenzidine) in concentrated acetic acid and optimal determination at 600 nm. (In this publication at 578 nm due to the use of a spectrophotometer with Hg-discharge lamp and filter). The appearance of a blue colour in the reaction tube or cuvette indicated the presence of haemoglobin. The use of the above chromogen, with its absorption maximum around 600 nm excluded interference from serum components such as bilirubin, which may interfere in the conventional method often used to determine plasma haemoglobin. The method can be used quantitatively by including an aqueous human haemoglobin standard in the run. This elutes from the HPLC column only as free haemoglobin in the concentration range from 0.1 to 10 g/l. Addition of human haemoglobin to haemoglobin-free plasma resulted in the binding of all Hb to plasma proteins up to a concentration between 2 and 3 g/l (void-volume fraction). At higher concentrations free Hb appeared in the 3-5 minute fractions. These observations agree with published data on the scavenging capacity of plasma for Hb released from erythrocytes. The method is rapid, (HPLC-run maximally 6 min, quantitative colorimetric results 5-10 min) precise (inter-assay coefficients of variation < 8%) and suitable for answering the question as to whether the protein-binding (scavenging) system which prevents the nephro- and cerebrotoxic effects of haemoglobin has been saturated or not, an important question in patients with acute haemolysis problems. A qualitative result is obtainable within 10 minutes of injecting the sample into the HPLC-system. The use of this assay in controlling blood transfusion and haemolytic events arising from surgery, intravascular haemolytic bacteria or artificial heart valves can help in rapid corrective action, if needed.

Acetic Acid↗

PubMed2000

Topical acetyl salicylate and dipyrone attenuate neurogenic protein extravasation in rat skin in vivo.

The effect of topically applied acetyl salicylic acid (ASA) and dipyrone on capsaicin-evoked protein extravasation was investigated by dermal microdialysis in rat. After a baseline of 75 min, capsaicin (1%) was applied epicutaneously under occlusion for 75 min above the capillaries. Topical capsaicin stimulation induced neurogenic protein extravasation with a mean increase of protein concentration in the perfusate of 165+/-27% (mean+/-SEM; n=15), whereas in sham-stimulated sites protein concentration decreased to 73+/-7% of the prestimulation value (n=6). ASA (2-200 mg/ml) and dipyrone (3-300 mg/ml) dose-dependently reduced the capsaicin induced protein extravasation to 118+/-23% (ASA, 200 mg/ml; n=8) and 72+/-9% (dipyrone, 300 mg/ml; n=8) of the prestimulation value. ASA and dipyrone antagonized the excitatory effects of capsaicin on skin nociceptors and thus suppressed the neurogenic protein extravasation.

Administration, Topical↗

PubMed2000

Involvement of the proinflammatory cytokines tumor necrosis factor-alpha, IL-1 beta, and IL-6 but not IL-8 in the development of heat hyperalgesia: effects on heat-evoked calcitonin gene-related peptide release from rat skin.

Proinflammatory cytokines contribute to the development of inflammatory and neuropathic pain and hyperalgesia in many in vivo models. The rat skin model was used to investigate the effects of proinflammatory cytokines on the basal and heat-evoked release of calcitonin gene-related peptide from nociceptors in vitro. In contrast to the excitatory effects of cytokines observed in vivo, none of the cytokines tested evoked any calcitonin gene-related peptide (CGRP) release at normal skin temperature of 32 degrees C. However, the cytokines IL-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 but not IL-8 induced a pronounced and transient sensitization of the heat-evoked CGRP release from nociceptors in vitro. This heat sensitization was dose dependent, with EC(50) for IL-1 beta of 2.7 ng/ml and for TNF-alpha of 3.1 ng/ml. The maximum IL-1 beta effect reached almost 600% of the heat-evoked release, and the maximum TNF-alpha effect induced a rise in CGRP release of 350%. In contrast to IL-1 beta and TNF-alpha, IL-6 did not induce heat sensitization when applied alone but was only effective in the presence of soluble IL-6 receptor. This suggests a constitutive expression of signaling receptors for TNF and IL-1 beta and the signal transduction molecule gp130 but not IL-6 receptor or IL-8 receptor. Furthermore, the acute cytokine signaling observed in the present study was independent of transcriptional pathways because sensitization occurred on short latency in vitro and under conditions that excluded chemotactic accumulation of immune cells from blood vessels. Our results demonstrate that interleukins may play an important role in the initiation of heat hyperalgesia in inflammation and neuropathy.

Animals↗

PubMed2000

Interactions of inflammatory mediators and low pH not influenced by capsazepine in rat cutaneous nociceptors.

The rat skin-saphenous nerve preparation was used to record from mechano-heat sensitive C-fibers whose receptive fields were superfused with various solutions of low pH and of bradykinin, serotonin and prostaglandin E2. Only synchronous application of protons and mediators resulted in a significant nearly three-fold augmentation of the nociceptive pH response, and capsazepine (10(-5) M) did not block this short-lived enhancement. Thus, it does not seem to involve the capsaicin receptor (VRI) which is in contrast to a previous finding from cultured sensory neurons.

Animals↗

PubMed2000

Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat.

The interactions between the inflammatory mediators bradykinin, serotonin, prostaglandin E(2) and acid pH were studied in rat dorsal root ganglion neurons in culture. For this purpose, the cultures were stimulated by inflammatory mediators (bradykinin, serotonin, prostaglandin E(2), 10(-5)M each) or acid solution (pH 6.1) for 5 min and the content of calcitonin gene-related peptide was determined in the supernatant before, during and after stimulation, using an enzyme immunoassay. Acid solution resulted in a threefold increase of the basal calcitonin gene-related peptide release which was entirely dependent on the presence of extracellular calcium. The release could not be blocked by the addition of the capsaicin antagonist capsazepine (10(-5)M). Bradykinin (10(-5)M) caused a 50% increase of the basal calcitonin gene-related peptide release which was again dependent on the presence of extracellular calcium, whereas serotonin and prostaglandin E(2) were each ineffective at 10(-5)M concentration. The combination of bradykinin, serotonin and prostaglandin E(2) led to a fivefold increase of the calcitonin gene-related peptide release which could not be further enhanced by acidification. The competitive capsaicin receptor antagonist capsazepine (10(-5)M) significantly reduced the release induced by the combination of bradykinin, serotonin and prostaglandin E(2). It is suggested that the inflammatory mediators co-operate and together may act as endogenous agonists at the capsaicin receptor to cause calcium influx and consecutive neuropeptide release.

Acids↗