Different Desensitization Patterns for Sensory and Vascular TRPV1 Populations in the Rat: Expression,Localization and Functional Consequences

Background and purpose

TRPV1 is expressed in sensory neurons and vascular smooth muscle cells, contributing to both pain perception and tissue blood distribution. Local desensitization of TRPV1 in sensory neurons by prolonged, high dose stimulation is re-engaged in clinical practice to achieve analgesia, but the effects of such treatments on the vascular TRPV1 are not known.

Experimental approach

Newborn rats were injected with capsaicin for five days. Sensory activation was measured by eye wiping tests and plasma extravasation. Isolated, pressurized skeletal muscle arterioles were used to characterize TRPV1 mediated vascular responses, while expression of TRPV1 was detected by immunohistochemistry.

Key results

Capsaicin evoked sensory responses, such as eye wiping (3.6±2.5 versus 15.5±1.4 wipes, p<0.01) or plasma extravasation (evans blue accumulation 10±3 versus 33±7 µg/g, p<0.05) were reduced in desensitized rats. In accordance, the number of TRPV1 positive sensory neurons in the dorsal root ganglia was also decreased. However, TRPV1 expression in smooth muscle cells was not affected by the treatment. There were no differences in the diameter (192±27 versus 194±8 µm), endothelium mediated dilations (evoked by acetylcholine), norepinephrine mediated constrictions, myogenic response and in the capsaicin evoked constrictions of arterioles isolated from skeletal muscle.

Conclusion and implications

Systemic capsaicin treatment of juvenile rats evokes anatomical and functional disappearance of the TRPV1-expressing neuronal cells but does not affect the TRPV1-expressing cells of the arterioles, implicating different effects of TRPV1 stimulation on the viability of these cell types.     

Stir-up effect of wind on a more-or-less stratified shallow lake phytoplankton community,Lake Balaton,Hungary

Microstratification of phytoplankton in the large shallow Lake Balaton (Hungary) was studied during a 24 h period. Dissolved O₂ showed biological stratification; flagellates exhibited a definite circadian rhythm. In the middle of the investigation a heavy storm broke out which was followed by the disappearance of differences between different layers of water. Storm-induced destratification is described by cluster-analysis. Abundances of dominant species changed differently in connection with the storm. Numbers of Nitzschia sp. increased due to stirring up from the sediment surface. Numbers of single-celled or colony-forming species (Cyclotella comta, Crucigenia quadrata, Coelosphaerium kuetzingianum) practically did not change. Numbers of all the three dominant filamentous species (Aphanizomenon fos-aquae f. klebahnii, Lyngbya limnetica, Planctonema lauterbornii) significantly decreased, which might be attributed to an unknown loss process and was followed by a competitive displacement by algae of small cell size.     

Dominant species, functional assemblages and frequency of equilibrium phases in late summer phytoplankton assemblages in Hungarian small shallow lakes

Late summer phytoplankton associations were studied qualitatively and quantitatively in 80 Hungarian lakes altogether (mostly shallow salt lakes, reservoirs, oxbows, gravel pit lakes). Equilibrium phases sensu Sommer et al. (1993) were found only in 17 lakes. Most of them were under some kind (high salt content or very low level of nutrients) of stress factor. It is concluded that environmental stress forces phytoplankton communities towards equilibrium. No relationship between occurrence of equilibria and trophic state was found. Species number of non-equilibrated lakes was almost three times as high as those in equilibrium. Of the 31 recently described (Reynolds et al., 2002) phytoplankton assemblages most of those were recognized that are likely to occur in shallow lakes. Separation of a functional group W _S from W2 for Synura dominated lakes is suggested. It seemed also necessary to raise a group (Y _Ph) for lakes dominated by Phacotus. Sorting of Dinophyta species into different already described functional groups is desirable.     

Changes in the expression of genes related to apoptosis and fibrosis pathways in CCl4-treated rats

Chronic liver diseases are accompanied by changes in the biochemical pathways related to the regulation of apoptosis and extra-cellular matrix deposition. The present study was designed to investigate, using low density arrays, changes in the hepatic gene expression together with hepatic biochemical and histological alterations in rats that had liver impairment induced by chronic exposure to CCl₄. Further, we examined the possible recovery of genetic and pathological changes following the cessation of the hepatotoxic injury. Experimental fibrosis was induced in male Wistar rats by CCl₄ administration. Animals were subdivided into two groups. One group was given CCl₄ and animals were killed at 8 and 12 weeks of treatment. The other group was treated with CCl₄ for 6 weeks, the CCl₄ was then stopped and, subsequently, subgroups of animals were killed after 1 and 2 weeks of recovery. CCl₄ administration over 12 weeks was associated with significant changes in B-cell leukemia/lymphoma 2, procollagen type I α 2, matrix metalloproteinases 3 and 8, tissue inhibitors of metalloproteinases 1, 2, and 3 and the inhibitor of apoptosis 4 gene expressions. Recovery after CCl₄ cessation was associated with changes in procollagen type I α 2, matrix metalloproteinase 7, tissue inhibitors of metalloproteinases 1 and 2, inhibitor of apoptosis 4, and survivin gene expressions. This study shows an association between changes in the expression of several genes regulating hepatic cell apoptosis, the fibrosis process, and the recovery of the hepatic function after removal of the toxic injury.     

mRNA transcription profile of potato (Solanum tuberosum L.) exposed to ultrasound during different stages of in vitro plantlet development

Plant Molecular Biology - In response to an ultrasound pulse, several hundred DEGs, including in response to stress, were up- or down-regulated in in vitro potato plantlets. Despite this abiotic...     

Cariprazine (RGH-188), a potent D3/D2 dopamine receptor partial agonist, binds to dopamine D3 receptors in vivo and shows antipsychotic-like and procognitive effects in rodents

We investigated the in vivo effects of orally administered cariprazine (RGH-188; trans-N-{4-[2-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-ethyl]-cyclohexyl}-N′,N′-dimethyl-urea), a D₃/D₂ dopamine receptor partial agonist with ∼10-fold preference for the D₃ receptor. Oral bioavailability of cariprazine at a dose of 1 mg/kg in rats was 52% with peak plasma concentrations of 91 ng/mL. Cariprazine 10 mg/kg had good blood-brain barrier penetration, with a brain/plasma AUC ratio of 7.6:1. In rats, cariprazine showed dose-dependent in vivo displacement of [³H](+)-PHNO, a dopamine D₃ receptor-preferring radiotracer, in the D₃ receptor-rich region of cerebellar lobules 9 and 10. Its potent inhibition of apomorphine-induced climbing in mice (ED₅₀ = 0.27 mg/kg) was sustained for 8 h. Cariprazine blocked amphetamine-induced hyperactivity (ED₅₀ = 0.12 mg/kg) and conditioned avoidance response (CAR) (ED₅₀ = 0.84 mg/kg) in rats, and inhibited the locomotor-stimulating effects of the noncompetitive NMDA antagonists MK-801 (ED₅₀ = 0.049 mg/kg) and phencyclidine (ED₅₀ = 0.09 mg/kg) in mice and rats, respectively. It reduced novelty-induced motor activity of mice (ED₅₀ = 0.11 mg/kg) and rats (ED₅₀ = 0.18 mg/kg) with a maximal effect of 70% in both species. Cariprazine produced no catalepsy in rats at up to 100-fold dose of its CAR inhibitory ED₅₀ value. Cariprazine 0.02-0.08 mg/kg significantly improved the learning performance of scopolamine-treated rats in a water-labyrinth learning paradigm. Though risperidone, olanzapine, and aripiprazole showed antipsychotic-like activity in many of these assays, they were less active against phencyclidine and more cataleptogenic than cariprazine, and had no significant effect in the learning task. The distinct in vivo profile of cariprazine may be due to its higher affinity and in vivo binding to D₃ receptors versus currently marketed typical and atypical antipsychotics.     

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [³H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([³H]Met-enkephalin-Arg⁶-Phe⁷), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K _D value of the radioligand calculated from homologous displacement experiments was 3.4 n M , and the maximal number of specific binding sites was 630 fmol/mg of protein. The K _D determined from equilibrium saturation binding studies was found to be 3.6 n M . However, the Hill coefficient was far below unity ( n _H = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [³H]Met-enkephalin-Arg⁶-Phe⁷ binding reflects a κ₂- and/or δ-subtype specificity of the heptapeptide. Binding to a κ₁ and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg⁶-Phe⁷ in frogs cannot be ruled out. The [³H]Met-enkephalin-Arg⁶-Phe⁷ binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.     

The effect of the CYP 2C19*2 polymorphism on stroke care

Clopidogrel is an inhibitor of platelet-aggregation used in the prevention of secondary stroke. The molecule is activated by the cytochrome P450 2C19 (CYP2C19) enzyme. The frequent CYP2C19*2 point mutation causes loss of enzyme function, a decreased (heterozygous form) or blocked (homozygous form) formation of the active molecule. Thus, for a patient harboring a mutated allele, clopidogrel does not provide effective protection against stroke. Multiple drugs inhibit the CYP2C19 enzyme and their simultaneous use with clopidogrel is especially hazardous for patients with genetically decreased enzyme activity. Frequency of the CYP2C19*2 is variable in different populations, highest rates were detected in some Asian groups. In our study the CYP2C19 genotype was determined in one Hungarian sample of 354 stroke patients and 221 healthy controls. Frequency of the minor allele was found to be 12.87% (12.85% in stroke patients, 12.89% in healthy controls). The proportion of the homozygous CYP2C19*2 variant causing total loss of gene function was 1.74%, rate of the heterozygous allele causing reduced enzyme activity was 22.26% in the total population. Our results for the allele frequencies of the CYP2C19*2 gene are similar to those found in other Caucasian populations. In conclusion, the homozygous mutation, causing ineffectiveness of clopidogrel is relatively rare. However, the heterozygous form in which interaction of CYP2C19 inhibitors causes further decrease in the genetically impaired enzyme activity is present in every fifth drug-taking patient. Based on our findings, we would like to emphasize that it is important to adjust individually antiplatelet treatment in ischemic stroke patients and to take into consideration genetic factors as well as drugs taken for comorbid conditions.     

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.