Annett Eitner

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor 2-mediated hepatocellular carcinoma cell invasion

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

Neuronal prostaglandin E2 receptor subtype EP3 mediates antinociception during inflammation

The pain mediator prostaglandin E2 (PGE2) sensitizes nociceptive pathways through EP2 and EP4 receptors, which are coupled to Gs proteins and increase cAMP. However, PGE2 also activates EP3 receptors, and the major signaling pathway of the EP3 receptor splice variants uses inhibition of cAMP synthesis via Gi proteins. This opposite effect raises the intriguing question of whether the Gi-protein–coupled EP3 receptor may counteract the EP2 and EP4 receptor-mediated pronociceptive effects of PGE2. We found extensive localization of the EP3 receptor in primary sensory neurons and the spinal cord. The selective activation of the EP3 receptor at these sites did not sensitize nociceptive neurons in healthy animals. In contrast, it produced profound analgesia and reduced responses of peripheral and spinal nociceptive neurons to noxious stimuli but only when the joint was inflamed. In isolated dorsal root ganglion neurons, EP3 receptor activation counteracted the sensitizing effect of PGE2, and stimulation of excitatory EP receptors promoted the expression of membrane-associated inhibitory EP3 receptor. We propose, therefore, that the EP3 receptor provides endogenous pain control and that selective activation of EP3 receptors may be a unique approach to reverse inflammatory pain. Importantly, we identified the EP3 receptor in the joint nerves of patients with painful osteoarthritis.

Effects of prostaglandin D2 on tetrodotoxin-resistant Na+ currents in DRG neurons of adult rat

&NA; Tetrodotoxin‐resistant (TTX‐R) Na+ channels play a key role in the generation of action potentials in nociceptive dorsal root ganglion (DRG) neurons and are an important target for the proinflammatory mediator prostaglandin E2, which augments these currents. Prostaglandin D2 (PGD2) is released in the tissue together with prostaglandin E2, and it was reported to be antiinflammatory, but its effect on primary afferent neurons is unclear. In the present study we localised Gs‐protein‐coupled DP1 and Gi‐protein‐coupled DP2 receptors in DRG neurons, and we assessed the effect of PGD2 on TTX‐R Na+ currents in patch‐clamp recordings from small‐ to medium‐sized cultured DRG neurons from adult rats. DP1 and DP2 receptor‐like immunoreactivity was localised in the vast majority of DRG neurons. In all neurons, PGD2 shifted conductance to more hyperpolarised potentials, depending on an action at Nav1.9 channels. In about one third of the neurons, PGD2 additionally influenced Nav1.8 channels by facilitating conductance and by increasing maximal current amplitudes. Selective DP1 receptor activation increased the amplitude of TTX‐R Na+ currents of most neurons, but this effect was counteracted by DP2 receptor activation, which by itself had no effect. In the current‐clamp mode, PGD2 lowered the threshold for elicitation of an action potential and increased the number of action potentials per stimulus, an effect mainly depending on DP1 receptor activation. Thus, the net effect of PGD2 on DRG neurons is pronociceptive, although the magnitude of the TTX‐R Na+ currents depends on the balance of DP1 and DP2 receptor activation. Prostaglandin D2 regulates conductance and current amplitudes of tetrodotoxin‐resistant sodium channels on dorsal root ganglion neurons by a balanced action at DP1 and DP2 receptor subtypes.

High-resolution two-photon excitation microscopy of ocular tissues in porcine eye

Two‐photon excitation laser scanning microscopy (TPM), based on nonlinear optical (NLO) response under high irradiance, is currently being extensively employed for diagnostic purposes in biomedical fields and becomes more and more an interesting imaging technique in the intact bulk tissue examination. In this study, this nonlinear‐excitation imaging technique including two‐photon‐mediated autofluorescence (2PF) and second harmonic generation (SHG) was employed to investigate the microstructures in the whole‐mount scleral, retinal, and corneal tissues of porcine eyes with intracellular spatial resolution and high signal‐to‐noise ratio.

Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo

Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF.

N,N-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry.

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol–ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.

Effects of Differently Activated Rodent Macrophages on Sensory Neurons: Implications for Arthritis Pain

In arthritis, macrophages invade the affected joint. Experimental arthritis models have shown that macrophages also invade the dorsal root ganglia (DRGs) of the inflamed segments in which the perikarya of sensory neurons are located. It is unclear whether this macrophage invasion contributes to arthritis pain and/or furthers neuronal damage. The present study was undertaken to investigate how differently activated macrophages affect DRG neurons.

Expression of corticosteroid binding globulin in the rat olfactory system.

Glucocorticoids are known to act on the olfactory system although their mode of action is still unclear since nuclear glucocorticoid receptors are mostly absent in the olfactory mucosa. In this study we used immunocytochemistry, in situ hybridization, and RT-PCR to study the expression and distribution of corticosteroid binding globulin (CBG) in the rat olfactory system. Mucosal goblet cells could be immunostained for CBG. Nasal secretion contained measurable amounts of CBG suggesting that CBG is liberated. CBG immunoreactivity was localized in many of the basal cells of the olfactory mucosa, while mature sensory cells contained CBG only in processes as determined by double immunostaining with the olfactory marker protein OMP. This staining was most pronounced in the vomeronasal organ (VNO). The appearance of CBG in the non-sensory and sensory parts of the VNO and in nerve terminals in the accessory bulb indicated axonal transport. Portions of the periglomerular cells, the mitral cells and the tufted cells were also CBG positive. CBG encoding transcripts were confirmed by RT-PCR in homogenates of the olfactory mucosa and VNO. Olfactory CBG may be significant for uptake, accumulation and transport of glucocorticoids, including aerosolic cortisol.

Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

During peripheral inflammation, both spinal TNF-α and IL-6 are released within the spinal cord and support the generation of inflammation-evoked spinal hyperexcitability. However, whether spinal TNF-α and IL-6 act independently in parallel or in a functionally dependent manner has not been investigated. In extracellular recordings from mechanonociceptive deep dorsal horn neurons of normal rats in vivo, we found that spinal application of TNF-α increased spinal neuronal responses to mechanical stimulation of knee and ankle joints. This effect was significantly attenuated by either sgp130, which blocks IL-6 trans-signaling mediated by IL-6 and its soluble receptor IL-6R (sIL-6R); by an antibody to the IL-6 receptor; or by minocycline, which inhibits the microglia. IL-6 was localized in neurons of the spinal cord and, upon peripheral noxious stimulation in the presence of spinal TNF-α, IL-6 was released spinally. Furthermore, TNF-α recruited microglial cells to provide sIL-6R, which can form complexes with IL-6. Spinal application of IL-6 plus sIL-6R, but not of IL-6 alone, enhanced spinal hyperexcitability similar to TNF-α and the inhibition of TNF-α-induced hyperexcitability by minocycline was overcome by coadministration of sIL-6R, showing that sIL-6R is required. Neither minocycline nor the TNF-α-neutralizing compound etanercept inhibited the induction of hyperexcitability by IL-6 plus sIL-6R. Together, these data show that the induction of hyperexcitability of nociceptive deep dorsal horn neurons by TNF-α largely depends on the formation of IL-6/sIL-6R complexes that are downstream of TNF-α and requires the interactions of neurons and microglia orchestrated by TNF-α. SIGNIFICANCE STATEMENT Both spinal TNF-α and IL-6 induce a state of spinal hyperexcitability. We present the novel finding that the full effect of TNF-α on the development of spinal hyperexcitability depends on IL-6 trans-signaling acting downstream of TNF-α. IL-6 trans-signaling requires the formation of complexes of IL-6 and soluble IL-6 receptor. Spinal TNF-α furthers the release of IL-6 from neurons in the spinal cord during peripheral noxious stimulation and recruits microglial cells to provide soluble IL-6 receptor, which can form complexes with IL-6. Therefore, a specific interaction between neurons and microglia is required for the full development of TNF-α-induced hyperexcitability of nociceptive deep horsal horn neurons.

Molecular effects of interleukin-1β on dorsal root ganglion neurons: Prevention of ligand-induced internalization of the bradykinin 2 receptor and downregulation of G protein-coupled receptor kinase 2

In dorsal root ganglion sections numerous small-to medium-sized neurons were found to exhibit extensive colocalization of the bradykinin receptor 2, the interleukin-1 receptor 1 and G protein-coupled receptor kinase 2. Application of bradykinin to cultured DRG neurons caused substantial internalization of the bradykinin 2 receptor which significantly reduced the responsiveness of DRG neurons to a second application of bradykinin. Such an internalization was not observed in DRG neurons which were exposed to long-term pretreatment with interleukin-1β. The long-term incubation with interleukin-1β on its own did neither change the proportion of neurons which expressed the bradykinin 2 receptor in the cytoplasma nor the proportion of neurons expressing the bradykinin 2 receptor in the membrane but it reduced the proportion of neurons expressing G protein-coupled receptor kinase 2, an enzyme which facilitates the internalization of G protein-coupled receptors. These results show that interleukin-1β maintains the responsiveness of DRG neurons to bradykinin in the long-term range, and they suggest that the downregulation of G protein-coupled receptor kinase 2 could be a cellular mechanism involved in this interleukin-1β effect.