PACAP-38 and PAC1 Receptor Alterations in Plasma and Cardiac Tissue Samples of Heart Failure Patients.

Pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) is a multifunctional neuropeptide, which may play a role in cardioprotection. However, little is known about the presence of PACAP-38 in heart failure (HF) patients. The aim of our study was to measure the alterations of PACAP-38 like immunoreactivity (LI) in acute (n = 13) and chronic HF (n = 33) and to examine potential correlations between PACAP-38 and HF predictors (cytokines, NT-proBNP). Tissue PACAP-38 LI and PAC1 receptor levels were also investigated in heart tissue samples of patients with HF. Significantly higher plasma PACAP-38 LI was detected in patients with acute HF, while in chronic HF patients, a lower level of immunoreactivity was observed compared to healthy controls (n = 13). Strong negative correlation was identified between plasma PACAP-38 and NT-proBNP levels in chronic HF, as opposed to the positive connection seen in the acute HF group. Plasma IL-1 ß, IL-2 and IL-4 levels were significantly lower in chronic HF, and IL-10 was significantly higher in patients with acute HF. PACAP-38 levels of myocardial tissues were lower in all end-stage HF patients and lower PAC1 receptor levels were detected in the primary dilated cardiomyopathy group compared to the controls. We conclude that PACAP-38 and PAC1 expression correlates with some biomarkers of acute and chronic HF; therefore, further studies are necessary to explore whether PACAP could be a suitable prognostic biomarker in HF patients.

PACAP-38 in Acute ST-Segment Elevation Myocardial Infarction in Humans and Pigs: A Translational Study.

Acute myocardial infarction (MI) is one of the most common causes of death worldwide. Pituitary adenylate cyclase activating polypeptide (PACAP) is a cardioprotective neuropeptide expressing its receptors in the cardiovascular system. The aim of our study was to examine tissue PACAP-38 in a translational porcine MI model and plasma PACAP-38 levels in patients with ST-segment elevation myocardial infarction (STEMI). Significantly lower PACAP-38 levels were detected in the non-ischemic region of the left ventricle (LV) in MI heart compared to the ischemic region of MI-LV and also to the Sham-operated LV in porcine MI model. In STEMI patients, plasma PACAP-38 level was significantly higher before percutaneous coronary intervention (PCI) compared to controls, and decreased after PCI. Significant negative correlation was found between plasma PACAP-38 and troponin levels. Furthermore, a significant effect was revealed between plasma PACAP-38, hypertension and HbA1c levels. This was the first study showing significant changes in cardiac tissue PACAP levels in a porcine MI model and plasma PACAP levels in STEMI patients. These results suggest that PACAP, due to its cardioprotective effects, may play a regulatory role in MI and could be a potential biomarker or drug target in MI.

Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins.

Ischaemic post-conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non-responsiveness to IPoC possibly caused by co-morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC-R) and non-responder (IPoC-NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre-conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT-PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC-R) or no protection (IPoC-NR). Twenty-one miRs were up-regulated by I/R and modified by IPoC. qRT-PCR analysis revealed that IPoC-R differed from other groups by reduced expression of arginase-2 and bax, whereas the mitochondrial uncoupling protein (UCP)-2 was induced in IPC and IPoC-R. IPoC-R and IPoC-NR synergistically increased the expression of non-mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium-handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC-NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC-R. This study underlines the importance of mitochondrial proteins for successful long-term outcome.

Cardiac miRNA Expression and their mRNA Targets in a Rat Model of Prediabetes.

Little is known about the mechanism of prediabetes-induced cardiac dysfunction. Therefore, we aimed to explore key molecular changes with transcriptomic and bioinformatics approaches in a prediabetes model showing heart failure with preserved ejection fraction phenotype. To induce prediabetes, Long-Evans rats were fed a high-fat diet for 21 weeks and treated with a single low-dose streptozotocin at week 4. Small RNA-sequencing, in silico microRNA (miRNA)-mRNA target prediction, Gene Ontology analysis, and target validation with qRT-PCR were performed in left ventricle samples. From the miRBase-annotated 752 mature miRNA sequences expression of 356 miRNAs was detectable. We identified two upregulated and three downregulated miRNAs in the prediabetic group. We predicted 445 mRNA targets of the five differentially expressed miRNAs and selected 11 mRNAs targeted by three differentially expressed miRNAs, out of which five mRNAs were selected for validation. Out of these five targets, downregulation of three mRNAs i.e., Juxtaposed with another zinc finger protein 1 (Jazf1); RAP2C, member of RAS oncogene family (Rap2c); and Zinc finger with KRAB and SCAN domains 1 (Zkscan1) were validated. This is the first demonstration that prediabetes alters cardiac miRNA expression profile. Predicted targets of differentially expressed miRNAs include Jazf1, Zkscan1, and Rap2c mRNAs. These transcriptomic changes may contribute to the diastolic dysfunction and may serve as drug targets.

Resolvin D1 and D2 Inhibit Transient Receptor Potential Vanilloid 1 and Ankyrin 1 Ion Channel Activation on Sensory Neurons via Lipid Raft Modification.

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons and regulate nociceptor and inflammatory functions. Resolvins are endogenous lipid mediators. Resolvin D1 (RvD1) is described as a selective inhibitor of TRPA1-related postoperative and inflammatory pain in mice acting on the G protein-coupled receptor DRV1/GPR32. Resolvin D2 (RvD2) is a very potent TRPV1 and TRPA1 inhibitor in DRG neurons, and decreases inflammatory pain in mice acting on the GPR18 receptor, via TRPV1/TRPA1-independent mechanisms. We provided evidence that resolvins inhibited neuropeptide release from the stimulated sensory nerve terminals by TRPV1 and TRPA1 activators capsaicin (CAPS) and allyl-isothiocyanate (AITC), respectively. We showed that RvD1 and RvD2 in nanomolar concentrations significantly decreased TRPV1 and TRPA1 activation on sensory neurons by fluorescent calcium imaging and inhibited the CAPS- and AITC-evoked 45Ca-uptake on TRPV1- and TRPA1-expressing CHO cells. Since CHO cells are unlikely to express resolvin receptors, resolvins are suggested to inhibit channel opening through surrounding lipid raft disruption. Here, we proved the ability of resolvins to alter the membrane polarity related to cholesterol composition by fluorescence spectroscopy. It is concluded that targeting lipid raft integrity can open novel peripheral analgesic opportunities by decreasing the activation of nociceptors.

Carboxamido steroids inhibit the opening properties of transient receptor potential ion channels by lipid raft modulation.

Transient Receptor Potential (TRP) cation channels, like the TRP Vanilloid 1 (TRPV1) and TRP Ankyrin 1 (TRPA1), are expressed on primary sensory neurons. These thermosensor channels play a role in pain processing. We have provided evidence previously that lipid raft disruption influenced the TRP channel activation, and a carboxamido-steroid compound (C1) inhibited TRPV1 activation. Therefore, our aim was to investigate whether this compound exerts its effect through lipid raft disruption and the steroid backbone (C3) or whether altered position of the carboxamido group (C2) influences the inhibitory action by measuring Ca2+ transients on isolated neurons and calcium-uptake on receptor-expressing CHO cells. Membrane cholesterol content was measured by filipin staining and membrane polarization by fluorescence spectroscopy. Both the percentage of responsive cells and the magnitude of the intracellular Ca2+ enhancement evoked by the TRPV1 agonist capsaicin were significantly inhibited after C1 and C2 incubation, but not after C3 administration. C1 was able to reduce other TRP channel activation as well. The compounds induced cholesterol depletion in CHO cells, but only C1 induced changes in membrane polarization. The inhibitory action of the compounds on TRP channel activation develops by lipid raft disruption, and the presence and the position of the carboxamido group is essential.

Analgesic effect of dimethyl trisulfide in mice is mediated by TRPA1 and sst4 receptors.

TRPA1 receptors are calcium-permeable ligand-gated channels expressed in primary sensory neurons and involved in inflammation and pain. Activation of these neurons might have analgesic effect. Suggested mechanism of analgesic effect mediated by TRPA1 activation is the release of somatostatin (SOM) and its action on sst4 receptors. In the present study analgesic effect of TRPA1 activation on primary sensory neurons by organic trisulfide compound dimethyl trisulfide (DMTS) presumably leading to SOM release was investigated. Opening of TRPA1 by DMTS in CHO cells was examined by patch-clamp and fluorescent Ca2+ detection. Ca2+ influx upon DMTS administration in trigeminal ganglion (TRG) neurons of TRPA1 receptor wild-type (WT) and knockout (KO) mice was detected by ratiometric Ca2+ imaging. SOM release from sensory nerves of murine skin was assessed by radioimmunoassay. Analgesic effect of DMTS in mild heat injury-induced mechanical hyperalgesia was examined by dynamic plantar aesthesiometry. Regulatory role of DMTS on deep body temperature (Tb) was measured by thermocouple thermometry with respirometry and by telemetric thermometry. DMTS produced TRPA1-mediated currents and elevated [Ca2+]i in CHO cells. Similar data were obtained in TRG neurons. DMTS released SOM from murine sensory neurons TRPA1-dependently. DMTS exerted analgesic effect mediated by TRPA1 and sst4 receptors. DMTS-evoked hypothermia and hypokinesis were attenuated in freely-moving TRPA1 KO animals. Our study has presented original evidence regarding analgesic action of DMTS which might be due to TRPA1-mediated SOM release from sensory neurons and activation of sst4 receptors. DMTS could be a novel analgesic drug candidate.

TRPA1 deficiency is protective in cuprizone-induced demyelination-A new target against oligodendrocyte apoptosis.

Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long-term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone-induced demyelination. This toxin-induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen-activated protein kinase pathways, as well as transcription factor c-Jun and a proapoptotic Bcl-2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca2+ concentration modulate astrocyte functions, and influence the pro or anti-apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166-2180.

Evidence for the role of lipid rafts and sphingomyelin in Ca2+-gating of Transient Receptor Potential channels in trigeminal sensory neurons and peripheral nerve terminals.

Transient Receptor Potential (TRP) cation channels, such as TRP Vanilloid 1 and TRP Ankyrin repeat domain 1 (TRPV1 and TRPA1) are nocisensors playing important role to signal pain. Two "melastatin" TRP receptors, like TRPM8 and TRPM3 are also expressed in a subgroup of primary sensory neurons. These channels serve as thermosensors with unique thermal sensitivity ranges and are activated also by several exogenous and endogenous chemical ligands inducing conformational changes from various allosteric ("multisteric") sites. We analysed the role of plasma membrane microdomains of lipid rafts on isolated trigeminal (TRG) neurons and TRPV1-expressing CHO cell line by measuring agonist-induced Ca2+ transients with ratiometric technique. Stimulation-evoked calcitonin gene related peptide (CGRP) release from sensory nerve endings of the isolated rat trachea by radioimmunoassay was also measured. Lipid rafts were disrupted by cleaving sphingomyelin (SM) with sphingomyelinase (SMase), cholesterol depletion with methyl ß-cyclodextrin (MCD) and ganglioside breakdown with myriocin. It has been revealed that intracellular Ca2+ increase responses evoked by the TRPV1 agonist capsaicin, the TRPA1 agonsits allyl isothiocyanate (AITC) and formaldehyde as well as the TRPM8 activator icilin were inhibited after SMase, MCD and myriocin incubation but the response to the TRPM3 agonist pregnenolon sulphate was not altered. Extracellular SMase treatment did not influence the thapsigargin-evoked Ca2+-release from intracellular stores. Besides the cell bodies, SMase also inhibited capsaicin- or AITC-evoked CGRP release from peripheral sensory nerve terminals, this provides the first evidence for the importance of lipid raft integrity in TRPV1 and TRPA1 gating on capsaicin-sensitive nerve terminals. SM metabolites, ceramide and sphingosine, did not influence TRPA1 and TRPV1 activation on TRG neurons, TRPV1-expressing CHO cell line, and nerve terminals. We suggest, that the hydrophobic interactions between TRP receptors and membrane lipid raft interfaces modulate the opening properties of these channels and therefore, targeting this interaction might be a promising tool for drug developmental purposes.

Saxagliptin Cardiotoxicity in Chronic Heart Failure: The Role of DPP4 in the Regulation of Neuropeptide Tone.

Dipeptidyl-peptidase-4 (DPP4) inhibitors are novel medicines for diabetes. The SAVOR-TIMI-53 clinical trial revealed increased heart-failure-associated hospitalization in saxagliptin-treated patients. Although this side effect could limit therapeutic use, the mechanism of this potential cardiotoxicity is unclear. We aimed to establish a cellular platform to investigate DPP4 inhibition and the role of its neuropeptide substrates substance P (SP) and neuropeptide Y (NPY), and to determine the expression of DDP4 and its neuropeptide substrates in the human heart. Western blot, radio-, enzyme-linked immuno-, and RNA scope assays were performed to investigate the expression of DPP4 and its substrates in human hearts. Calcein-based viability measurements and scratch assays were used to test the potential toxicity of DPP4 inhibitors. Cardiac expression of DPP4 and NPY decreased in heart failure patients. In human hearts, DPP4 mRNA is detectable mainly in cardiomyocytes and endothelium. Treatment with DPP4 inhibitors alone/in combination with neuropeptides did not affect viability but in scratch assays neuropeptides decreased, while saxagliptin co-administration increased fibroblast migration in isolated neonatal rat cardiomyocyte-fibroblast co-culture. Decreased DPP4 activity takes part in the pathophysiology of end-stage heart failure. DPP4 compensates against the elevated sympathetic activity and altered neuropeptide tone. Its inhibition decreases this adaptive mechanism, thereby exacerbating myocardial damage.

Protective effects of the novel amine-oxidase inhibitor multi-target drug SZV 1287 on streptozotocin-induced beta cell damage and diabetic complications in rats.

Diabetes mellitus is a common metabolic disease leading to hyperglycemia due to insufficient pancreatic insulin production or effect. Amine oxidase copper containing 3 (AOC3) is an enzyme that belongs to the semicarbazide-sensitive amine oxidase family, which may be a novel therapeutic target to treat diabetic complications. We aimed to explore the effects of AOC3 inhibition and to test the actions of our novel AOC3 inhibitor multi-target drug candidate, SZV 1287, compared to a selective reference compound, LJP 1207, in an 8-week long insulin-controlled streptozotocin (STZ)-induced (60 mg/kg i.p.) rat diabetes model. Both AOC3 inhibitors (20 mg/kg, daily s.c. injections) were protective against STZ-induced pancreatic beta cell damage determined by insulin immunohistochemistry and radioimmunoassay, neuropathic cold hypersensitivity measured by paw withdrawal latency decrease from 0 °C water, and retinal dysfunction detected by electroretinography. SZV 1287 showed greater inhibitory effects on beta cell damage, and reduced retinal apoptosis shown by histochemistry. Mechanical hypersensitivity measured by aesthesiometry, cardiac dysfunction and nitrosative stress determined by echocardiography and immunohistochemistry/Western blot, respectively, serum Na+, K+, fructosamine, and urine microalbumin, creatinine, total protein/creatinine ratio alterations did not develop in response to diabetes. None of these parameters were influenced by the treatments except for SZV 1287 reducing serum fructosamine and LJP 1207 increasing urine creatinine. We provide the first evidence for protective effects of AOC3 inhibition on STZ-induced pancreatic beta cell damage, neuropathic cold hypersensitivity and diabetic retinal dysfunction. Long-term treatment with our novel multi-target analgesic candidate, SZV 1287, is safe and effective also under diabetic conditions.

Somatostatin and Its Receptors in Myocardial Ischemia/Reperfusion Injury and Cardioprotection.

Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles. SSTR1 and SSTR2, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated SSTR1 mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy. SSTR1, SSTR2, and SSTR5 but not SST and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples, SSTR1 and SSTR2 mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope® in situ hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded.

Analgesic Effects of Lipid Raft Disruption by Sphingomyelinase and Myriocin via Transient Receptor Potential Vanilloid 1 and Transient Receptor Potential Ankyrin 1 Ion Channel Modulation.

Transient Receptor Potential (TRP) Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons, and integratively regulate nociceptor and inflammatory functions. Lipid rafts are liquid-ordered plasma membrane microdomains rich in cholesterol, sphingomyelin and gangliosides. We earlier showed that lipid raft disruption inhibits TRPV1 and TRPA1 functions in primary sensory neuronal cultures. Here we investigated the effects of sphingomyelinase (SMase) cleaving membrane sphingomyelin and myriocin (Myr) prohibiting sphingolipid synthesis in mouse pain models of different mechanisms. SMase (50 mU) or Myr (1 mM) pretreatment significantly decreased TRPV1 activation (capsaicin)-induced nocifensive eye-wiping movements by 37 and 41%, respectively. Intraplantar pretreatment by both compounds significantly diminished TRPV1 stimulation (resiniferatoxin)-evoked thermal allodynia developing mainly by peripheral sensitization. SMase (50 mU) also decreased mechanical hyperalgesia related to both peripheral and central sensitizations. SMase (50 mU) significantly reduced TRPA1 activation (formalin)-induced acute nocifensive behaviors by 64% in the second, neurogenic inflammatory phase. Myr, but not SMase altered the plasma membrane polarity related to the cholesterol composition as shown by fluorescence spectroscopy. These are the first in vivo results showing that sphingolipids play a key role in lipid raft integrity around nociceptive TRP channels, their activation and pain sensation. It is concluded that local SMase administration might open novel perspective for analgesic therapy.

Hidden Cardiotoxicity of Rofecoxib Can be Revealed in Experimental Models of Ischemia/Reperfusion.

Cardiac adverse effects are among the leading causes of the discontinuation of clinical trials and the withdrawal of drugs from the market. The novel concept of 'hidden cardiotoxicity' is defined as cardiotoxicity of a drug that manifests in the diseased (e.g. ischemic/reperfused), but not in the healthy heart or as a drug-induced deterioration of cardiac stress adaptation (e.g. ischemic conditioning). Here, we aimed to test if the cardiotoxicity of a selective COX-2 inhibitor rofecoxib that was revealed during its clinical use, i.e., increased occurrence of proarrhythmic and thrombotic events, could have been revealed in early phases of drug development by using preclinical models of ischemia/reperfusion (I/R) injury. Rats that were treated with rofecoxib or vehicle for four weeks were subjected to 30 min. coronary artery occlusion and 120 min. reperfusion with or without cardioprotection that is induced by ischemic preconditioning (IPC). Rofecoxib increased overall the arrhythmias including ventricular fibrillation (VF) during I/R. The proarrhythmic effect of rofecoxib during I/R was not observed in the IPC group. Rofecoxib prolonged the action potential duration (APD) in isolated papillary muscles, which was not seen in the simulated IPC group. Interestingly, while showing hidden cardiotoxicity manifested as a proarrhythmic effect during I/R, rofecoxib decreased the infarct size and increased the survival of adult rat cardiac myocytes that were subjected to simulated I/R injury. This is the first demonstration that rofecoxib increased acute mortality due to its proarrhythmic effect via increased APD during I/R. Rofecoxib did not interfere with the cardiprotective effect of IPC; moreover, IPC was able to protect against rofecoxib-induced hidden cardiotoxicity. These results show that cardiac safety testing with simple preclinical models of I/R injury uncovers hidden cardiotoxicity of rofecoxib and might reveal the hidden cardiotoxicity of other drugs.

Targeted single-cell electroporation loading of Ca2+ indicators in the mature hemicochlea preparation.

Ca2+ is an important intracellular messenger and regulator in both physiological and pathophysiological mechanisms in the hearing organ. Investigation of cellular Ca2+ homeostasis in the mature cochlea is hampered by the special anatomy and high vulnerability of the organ. A quick, straightforward and reliable Ca2+ imaging method with high spatial and temporal resolution in the mature organ of Corti is missing. Cell cultures or isolated cells do not preserve the special microenvironment and intercellular communication, while cochlear explants are excised from only a restricted portion of the organ of Corti and usually from neonatal pre-hearing murines. The hemicochlea, prepared from hearing mice allows tonotopic experimental approach on the radial perspective in the basal, middle and apical turns of the organ. We used the preparation recently for functional imaging in supporting cells of the organ of Corti after bulk loading of the Ca2+ indicator. However, bulk loading takes long time, is variable and non-selective, and causes the accumulation of the indicator in the extracellular space. In this study we show the improved labeling of supporting cells of the organ of Corti by targeted single-cell electroporation in mature mouse hemicochlea. Single-cell electroporation proved to be a reliable way of reducing the duration and variability of loading and allowed subcellular Ca2+ imaging by increasing the signal-to-noise ratio, while cell viability was retained during the experiments. We demonstrated the applicability of the method by measuring the effect of purinergic, TRPA1, TRPV1 and ACh receptor stimulation on intracellular Ca2+ concentration at the cellular and subcellular level. In agreement with previous results, ATP evoked reversible and repeatable Ca2+ transients in Deiters', Hensen's and Claudius' cells. TRPA1 and TRPV1 stimulation by AITC and capsaicin, respectively, failed to induce any Ca2+ response in the supporting cells, except in a single Hensen's cell in which AITC evoked transients with smaller amplitude. AITC also caused the displacement of the tissue. Carbachol, agonist of ACh receptors induced Ca2+ transients in about a third of Deiters' and fifth of Hensen's cells. Here we have presented a fast and cell-specific indicator loading method allowing subcellular functional Ca2+ imaging in supporting cells of the organ of Corti in the mature hemicochlea preparation, thus providing a straightforward tool for deciphering the poorly understood regulation of Ca2+ homeostasis in these cells.

Behavioural alterations and morphological changes are attenuated by the lack of TRPA1 receptors in the cuprizone-induced demyelination model in mice.

We have recently reported that the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. The aim of the present study was to gather additional data on the role of TRPA1 by investigating the time course of behavioural alterations and morphological changes in cuprizone-treated TRPA1 receptor gene-deficient mice. Demyelination was induced by feeding male wild-type (WT) and TRPA1 gene-deleted (TRPA1 KO) mice with 0.2% cuprizone for 6 weeks. Behavioural tests were performed once per week to follow cuprizone-induced functional changes. Mechanonociceptive thresholds were investigated by a dynamic plantar aesthesiometer and von Frey filaments. Motor performance was assessed by accelerating RotaRod and horizontal grid tests. For the study of spontaneous activity, the open field test was used. The time course of corpus callosum demyelination was also followed weekly by magnetic resonance imaging (MRI). Histological analysis of myelin loss was performed with Luxol Fast Blue (LFB) staining at week 3 and electron microscopy (EM) at week 6. Astrocyte and microglia accumulation at week 3 was assessed by immunohistochemistry (IHC). Cuprizone treatment induced no changes in mechanonociception or motor performance. In the open arena, cuprizone-treated mice spent more time with locomotion, their mean velocity was significantly higher and the distance they travelled was longer than untreated mice. No statistical difference was detected between WT and TRPA1 KO mice in these parameters. On the other hand, significantly increased rearing behaviour was induced in WT mice compared to TRPA1 KO animals. Morphological changes detected with MRI, LFB, IHC and EM analysis revealed reduced damage of the myelin and attenuated accumulation of astrocytes and microglia in cuprizone-treated TRPA1 KO animals, at each examined time point. Our recent data further suggest that inhibition of TRPA1 receptors could be a promising therapeutic approach to limit central nervous system damage in demyelinating diseases.

Estradiol Sensitizes the Transient Receptor Potential Vanilloid 1 Receptor in Pain Responses.

Sex differences exist in chronic pain pathologies, and gonadal estradiol (E2) alters the pain sensation. The nocisensor transient receptor potential vanilloid 1 (TRPV1) receptor plays a critical role in triggering pain. Here we examined the impact of E2 on the function of TRPV1 receptor in mice sensory neurons in vitro and in vivo. Both mechano- and thermonociceptive thresholds of the plantar surface of the paw of female mice were significantly lower in proestrus compared with the estrus phase. These thresholds were higher in ovariectomized (OVX) mice and significantly lower in sham-operated mice in proestrus compared with the sham-operated mice in estrus phase. This difference was absent in TRPV1 receptor-deficient mice. Furthermore, E2 potentiated the TRPV1 receptor activation-induced mechanical hyperalgesia in OVX mice. Long pretreatment (14 hours) with E2 induced a significant increase in TRPV1 receptor messenger RNA expression and abolished the capsaicin-induced TRPV1 receptor desensitization in primary sensory neurons. The short E2 incubation (10 minutes) also prevented the desensitization, which reverted after coadministration of E2 and the tropomyosin-related kinase A (TrkA) receptor inhibitor. Our study provides in vivo and in vitro evidence for E2-induced TRPV1 receptor upregulation and sensitization mediated by TrkAR via E2-induced genomic and nongenomic mechanisms. The sensitization and upregulation of TRPV1 receptor by E2 in sensory neurons may explain the greater pain sensitivity in female mice.

Capsaicin-Sensitive Sensory Nerves Mediate the Cellular and Microvascular Effects of H2S via TRPA1 Receptor Activation and Neuropeptide Release.

It is supposed that TRPA1 receptor can be activated by hydrogen sulphide (H2S). Here, we have investigated the role of TRPA1 receptor in H2S-induced [Ca(2+)]i increase in trigeminal ganglia (TRG) neurons, and the involvement of capsaicin-sensitive sensory nerves in H2S-evoked cutaneous vasodilatation. [Ca(2+)]i was measured with ratiometric technique on TRG neurons of TRPA1(+/+) and TRPA1(-/-) mice after NaHS, Na2S, allylisothiocyanate (AITC) or KCl treatment. Microcirculatory changes in the ear were detected by laser Doppler imaging in response to topical NaHS, AITC, NaOH, NaSO3 or NaCl. Mice were either treated with resiniferatoxin (RTX), or CGRP antagonist BIBN4096, or NK1 receptor antagonist CP99994, or K(+) ATP channel blocker glibenclamide. Alpha-CGRP(-/-) and NK1 (-/-) mice were also investigated. NaHS and Na2S increased [Ca(2+)]i in TRG neurons derived from TRPA(+/+) but not from TRPA1(-/-) mice. NaHS increased cutaneous blood flow, while NaOH, NaSO3 and NaCl did not cause significant changes. NaHS-induced vasodilatation was reduced in RTX-treated animals, as well as by pre-treatment with BIBN4096 or CP99994 alone or in combination. NaHS-induced vasodilatation was significantly smaller in alpha-CGRP(-/-) or NK1 (-/-) mice compared to wild-types. H2S activates capsaicin-sensitive sensory nerves through TRPA1 receptors and the resultant vasodilatation is mediated by the release of vasoactive sensory neuropeptides CGRP and substance P.

Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17ß-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation in the rat endometrium in correlation with the MIF.

Synthesis of novel 13α-18-norandrostane-ferrocene conjugates via homogeneous catalytic methods and their investigation on TRPV1 receptor activation.

13α-Steroid-ferrocene derivatives were synthesized via two reaction pathways starting from an unnatural 16-keto-18-nor-13α-steroid. The unnatural steroid was converted to ferrocene derivatives via copper-catalyzed azide-alkyne cycloaddition or palladium-catalyzed aminocarbonylation. 16-Azido- and 16-N-(prop-2-ynyl)-carboxamido-steroids were synthesized as starting materials for azide-alkyne cycloaddition with the appropriate ferrocene derivatives. Based on our earlier work, aminocarbonylation of 16-iodo-16-ene and 16-iodo-15-ene derivatives was studied with ferrocenylmethylamine. The new products were obtained in moderate to good yields and were characterized by (1)H and (13)C NMR, IR and MS. The solid state structure of the starting material 13α-18-norandrostan-16-one and two carboxamide products were determined by X-ray crystallography. Evidences were provided that the N-propargyl-carboxamide compound as well as its ferrocenylmethyltriazole derivative are able to decrease the activation of TRPV1 receptor on TRG neurons.