Effects of butyrate− on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm2 with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH4+, K+, and Na+ and highly sensitive to GSK1016790A, while effects of butyrate− were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate− (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate− stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4)

The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed

The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia

Klinische, histopathologische und genetische Charakterisierung der myofibrillären Myopathien

INTRODUCION: Myofibrillar Myopathies (MFM) are a group of rare neuromuscular disorders with heterogeneous genetic and clinical findings. The inheritance is mainly autosomal dominant or sporadic. Hallmarks in histopathology are myofibrillar disorganization and accumulation of myofibrillar degradation-products. The aim of this study is the clinical, histopathological and genetic characterization of a group of MFM cases. METHODS AND CASES: These cases were selected by searching our clinico-pathological database. We studied the histopathological features of the myofibrillar myopathy cases or families in our neuromuscular biopsy archive (Institute of Neuropathology, RWTH University Hospital Aachen). General light microscopic techniques as well as immunohistochemistry and electron microscopy were used as morphological approaches. Next, we performed in these MFM patients a thorough characterization of the clinical and paraclinical phenotype: serum-CK, EMG, ENG, cardio-pulmonic findings and molecular-genetic screening (DES, MYOT, FLNC, ZASP, BAG3, CRYAB, FHL1). In 7 cases we performed Laser-Microdissection (LCM) and proteomic-analysis.RESULTS: We isolated 50 patients out of 15.500 cases (0.3%) with clinical and histopathological findings conformable with MFM. We studied the histopathological and clinical characteristics in our group and compared them to those referred in the literature. Finally we could confirm these hallmarks in our group. MFM is also in our group a rare disorder, most mutations were found in the DES, MYOT und FLNC gen. In 36 cases we performed genetic screening; in 11 cases a mutation was found (5 DES, 3 FLNC and 3 MYOT). One case showed a polymorphism in the ZASP-gen; in 14 cases all known mutations could be excluded, in 10 cases final genetic results were still ongoing. 8 of 35 cases showed a positive family-history for neuromuscular disorders, we have ascertained detailed pedigrees in 4 families. It was possible to perform LCM and proteomic-analysis in 7 cases; we found different protein-ratios and increased appearing of different proteins within the aggregates. DISCUSSION: In all of our cases we could proof the hallmarks of MFM which were referred in the literature. That supports the presumption of a consistent histopathological definition of MFM, even if there are heterogeneous clinical und genetic findings. Subform-characteristic changes are: in Desmin-mutations increased granulofilamentous accumulations; in Myotilin- and ZASP-Mutations show more severe z-disc-changes. BAG3-Mutations are associated with changes in the nucleus. Mutations in the FilaminC-Gen lead to more severe myofibrillar changes than the other MFM-forms. In the LMD and Proteomics-Analysis we could proof, that the isolated aggregates are consist of a deviating protein-pattern, and that special proteins are increased within the aggregates. The wide range of clinical phenotypes was also present in our group. Patients with Desminopathy showed the highest rate of critical cardiac complications, patients with Filaminopathy are often suffering from pulmonic complications. Patients with Myotilinopathy showed a very heterogeneous mix of symptoms

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed

Original recordings of two bTRPV3 cells filled with NMDG-gluconate solution.

<p>a) As before, replacement of chloride by gluconate resulted in a decrease in outward current at positive potentials, suggesting expression of chloride channels. Both addition of Ca²⁺ (10 mmol∙l⁻¹) and replacement by Ca-gluconate₂ (60 mmol∙l⁻¹) had a strong blocking effect, with washout to levels higher than before. The current could be partially blocked by Mg²⁺, arguing against a rupture of the seal. Note the current kinetics with amplitude decreasing after a hyperpolarising pulse suggesting that Mg²⁺ is being drawn into the channel pore by the negative membrane voltage. Conversely, a depolarising voltage pulse leads to a relief of the block. In both cases, the currents appear curved in contrast to the linear kinetics of currents in pure NMDG-gluconate solution. b) Second cell, showing a spontaneous activation of inward and outward current amplitude by a factor of about four between the first set of pulses in NMDG-gluconate EGTA solution (1) and a second set (2) that was run 100 seconds later. Current amplitude continued to increase both after addition of 10 mmol∙l⁻¹ Ca²⁺ to the NMDG-gluconate solution, and after replacement of NMDG-gluconate by Ca-gluconate₂. Both the current kinetics and the tail currents indicate a partial voltage-dependent block. The arrows above the first set of pulses indicate the interval used for the determination of mean current amplitude for the current-voltage plot, which is depicted in c), showing rectification as a further sign of a voltage-dependent block. The reversal potential in 60 mmol∙l⁻¹ Ca-gluconate₂ solution was 7.1 mV, revealing permeability to Ca²⁺.</p

Current-voltage plots of whole cell measurements from Fig 2.

<p>The figures give the mean current values (± SEM) of control cells (a, c) and cells expressing bTRPV3 (b, d) in Na-gluconate (a, b) or NH₄-gluconate (c, d) bath solutions with (●) and without (○) divalent cations, as indicated. (*: p < 0.05, tested for −100 mV and +100 mV).</p

Effects of menthol and Mg²⁺ on intracellular calcium [Ca²⁺]_i.

<p>a) Average of ten original recordings of control HEK-293 cells, showing a significant increase of Δ [Ca²⁺]_i in the 200 s interval after application of menthol (1 mmol∙l⁻¹) (95% confidence interval in grey), with the timeline identical to that in b), showing the average [Ca²⁺]_i (± 95%) of eleven recordings of HEK-293 cells overexpressing bTRPV3. While resting levels of [Ca²⁺]_i in bTRPV3 cells were not significantly different from those of control cells, menthol led to a significantly higher Δ [Ca²⁺]_i in these cells (p = 0.003). c) Boxplots comparing the slopes (Δ [Ca²⁺]_i /Δ t) of the graphs in (a and b) in a 50 s interval at the beginning of the measurement and in the 50 s interval after application of menthol (differing letters above the boxes indicate p < 0.05). d) Average [Ca²⁺]_i (± 95%) of further ten recordings of HEK-293 cells overexpressing bTRPV3. Application of 2, 5, and 10 mmol∙l⁻¹ Mg²⁺ induced a transient drop in [Ca²⁺]_i.</p

Original recordings of one patch from a bTRPV3 cell with NH₄Cl in the pipette.

<p>a) Channel events are clearly visible in NaCl bath solution in response to pulse protocol III both at negative potentials and positive potentials, reflecting influx of Na⁺ and efflux of NH₄⁺. b) same patch in 72.5 mmol∙l⁻¹ CaCl₂ bath solution. Ca²⁺ had a clear blocking effect on the current level at positive potentials (efflux of NH₄⁺). c) Detail from (b), showing single-channel events at negative potentials, reflecting influx of Ca²⁺ d) same patch in NMDGCl bath solution. e) Insert showing single-channel events at negative potential level, suggesting influx of NMDG⁺. f) Current-voltage plot of unitary current amplitudes from this individual patch, fitted with the current formulation of the Goldman-Hodgkin-Katz equation. The fit yields a conductance for Na⁺ of 132 ± 4 pS, for Ca²⁺ of 21 ± 3 pS, and for NMDG⁺ of 36 ± 3 pS. The conductance for NH₄⁺ was similar in NaCl and NMDGCl solution (250 ± 3 pS or 264 ± 3 pS, respectively) but dropped to 88 ± 4 pS with high Ca²⁺ in the bath.</p

Whole cell measurements of HEK-293 cells expressing bTRPV3.

<p>Cells were filled with a Na-gluconate pipette solution (a) and superfused with various bath solutions as indicated. Cells were stimulated via consecutive application of the continuous pulse protocol I (b), subsequently merged. This yielded traces such as that in (c), which represents the original recording of one individual bTRPV3 cell. At regular intervals (66 s), pulse protocol I was briefly interrupted to run the high resolution classical step protocol II shown in (d), yielding current responses such as that shown in (e).</p