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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.

SUMMARY As nanoparticles (NPs) are readily absorbed across gastrointestinal tract, it is hypothesized that Zinc oxide nanoparticles (ZnO-NPs) will not interfere with intestinal glucose transport. The current study aims to investigate the in vitro effects of ZnO-NPs on Na linked glucose transport across jejunal mucosa in laying hens. A total of 12 White Leghorn laying hens (1.73 ± 0.176 kg), 40 wk of age, housed in individual cages were killed and mid-jejunum was divided into 4 segments to be mounted on the Ussing chambers. The mucosal side of jejunum was bathed with either no ZnO, ZnO (70 μM), ZnO-NPs (70 μM), or ZnO-NPs (35 μM) followed by addition of D-glucose (10 mM) mucosally in all the 4 experimental groups. Peak electrical response was measured 2 min after the addition of D-glucose. Results demonstrate that there was a decrease (P

Aim: Absorption of ammonia from the gut has consequences that range from encephalitis in hepatic disease to global climate change induced by nitrogenous excretions from livestock. Since patch clamp data show that certain members of the transient receptor potential (TRP) family are permeable to NH4+, participation in ammonium efflux was investigated. Methods: Digesta, mucosa and muscular samples from stomach, duodenum, jejunum, ileum, caecum and colon of pigs were analysed via colourimetry, qPCR, Western blot, immunohistochemistry and Ussing chambers. Results: qPCR data show high duodenal expression of TRPV6. TRPM6 was highest in jejunum and colon, with expression of TRPM7 ubiquitous. TRPM8 and TRPV1 were below detection. TRPV2 was highest in the jejunum but almost non-detectable in the colon. TRPV4 was ubiquitously expressed by mucosal and muscular layers. TRPV3 mRNA was only found in the mucosa of the caecum and colon, organs in which NH4+ was highest (>7 mmol·L−1). Immunohistochemically, an apical expression of TRPV3 and TRPV4 could be detected in all tissues, with effects of 2-APB and GSK106790A supporting functional expression. In symmetrical NaCl Ringer, removal of mucosal Ca2+ and Mg2+ increased colonic short circuit current (Isc) and conductance (Gt) by 0.18 ± 0.06 µeq·cm−2·h−1 and 4.70 ± 0.85 mS·cm−2 (P < .05, N/n = 4/17). Application of mucosal NH4Cl led to dose-dependent and divalent-sensitive increases in Gt and Isc, with effects highest in the caecum and colon. Conclusion: We propose that TRP channels contribute to the intestinal transport of ammonium, with TRPV3 and TRPV4 promising candidate proteins. Pharmacological regulation may be possible.

Magnesium (Mg2+) is an essential mineral without known specific regulatory mechanisms. In ruminants, plasma Mg2+concentration depends primarily on the balance between Mg2+absorption and Mg2+excretion. The primary site of Mg2+absorption is the rumen, where Mg2+is apically absorbed by both potential-dependent and potential-independent uptake mechanisms, reflecting involvement of ion channels and electroneutral transporters, respectively. Transport is energised in a secondary active manner by a basolateral Na+/Mg2+exchanger. Ruminal transport of Mg2+is significantly influenced by a variety of factors such as high K+concentration, sudden increases of ammonia, pH, and the concentration of SCFA. Impaired Mg2+absorption in the rumen is not compensated for by increased transport in the small or large intestine. While renal excretion can be adjusted to compensate precisely for any surplus in Mg2+uptake, a shortage in dietary Mg2+cannot be compensated for either via skeletal mobilisation of Mg2+or via up-regulation of ruminal absorption. In such situations, hypomagnesaemia will lead to decrease of a Mg2+in the cerebrospinal fluid and clinical manifestations of tetany. Improved knowledge concerning the factors governing Mg2+homeostasis will allow reliable recommendations for an adequate Mg2+intake and for the avoidance of possible disturbances. Future research should clarify the molecular identity of the suggested Mg2+transport proteins and the regulatory mechanisms controlling renal Mg excretion as parameters influencing Mg2+homeostasis.

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.

Results of recent in vitro experiments suggest that essential oils (EO) may not only influence ruminal fermentation but also modulate the absorption of cations like Na+, Ca2+ and NH4 + across ruminal epithelia of cattle and sheep through direct interaction with epithelial transport proteins, such as those of the transient receptor potential family. The aim of the current study was to examine this hypothesis by testing the effect of a blend of essential oils (BEO) on cation status and feed efficiency in lactating dairy cows. In the experiment, 72 dairy cows in mid-to-end lactation were divided into two groups of 36 animals each and fed the same mixed ration with or without addition of BEO in a 2×2 cross-over design. Feed intake, milk yield and composition, plasma and urine samples were monitored. Feeding BEO elevated milk yield, milk fat and protein yield as well as feed efficiency, whereas urea levels in plasma and milk decreased. In addition, plasma calcium levels increased significantly upon BEO supplementation, supporting the hypothesis that enhanced cation absorption might contribute to the beneficial effects of these EO.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

In ruminants, gastrointestinal recycling of urea is acutely enhanced by fibre-rich diets that lead to high ruminal concentration of short chain fatty acids (SCFA), while high ammonia has inhibitory effects. This study attempted to clarify if urea flux to the porcine cecum is similarly regulated. Thirty-two weaned piglets were fed diets containing protein (P) of poor prececal digestibility and fibre (F) at high (H) or low levels (L) in a 2 × 2 factorial design. After slaughter, cecal content was analyzed and the cecal mucosa incubated in Ussing chambers to measure the effect of pH, SCFA and NH4 + on the flux rates of urea, short-circuit current (I sc) and tissue conductance (G t). NH4 + significantly enhanced I sc (from 0.5 ± 0.2 to 1.2 ± 0.1 μEq cm−2 h−1). No acute effects of SCFA or ammonia on urea flux were observed. Tissue conductance was significantly lower in the high dietary fibre groups irrespective of the protein content. Only the HP-LF group emerged as different from all others in terms of urea flux (74 ± 6 versus 53 ± 3 nmol cm−2 h−1), associated with higher cecal ammonia concentration and reduced fecal consistency. The data suggest that as in the rumen, uptake of ammonia by the cecum may involve electrogenic transport of the ionic form (NH4 +). In contrast to findings in the rumen, neither a high fibre diet nor acute addition of SCFA enhanced urea transport across the pig cecum. Instead, a HP-LF diet had stimulatory effects. A potential role for urea recycling in stabilizing luminal pH is discussed.

Transition is defined as the period from 3 weeks before parturition until 3 weeks postpartum and is characterized by changes in endocrine status and by major alterations in the requirement for nutrients, vitamins and minerals. At parturition, the dry period diet is replaced by an ad libitum offered lactation diet, which causes changes of rumen fermentation because of its higher energy density and the overall increase of dry matter intake. Hence, ruminal fermentation parameters such as pH, volatile fatty acid (VFA) concentrations and osmotic pressure are changed. This causes alterations in the rumen epithelium generally termed adaptation: the modulation of enzyme activities, an increase in the number and size of papillae and enhanced transport rates of ions. These effects are well documented when sheep or cows are experimentally and independent of parturition shifted from a low energy (roughage) to a concentrate diet. Only a few studies have been conducted in cows during transition and, thus, data on diet dependent effects on postpartum rumen acidosis are absent and information regarding number and size of papillae is inconsistent. Furthermore, investigations of gene expression related to VFA absorption or metabolism have not shown diet dependent alterations. However, evidence from in vitro and in vivo studies exists that the stimulation of fermentation before parturition enhances the transport rates of Na, Cl and VFA across bovine rumen epithelium. The underlying mechanisms for epithelial adaptation to dietary alterations are poorly understood at present. Luminal factors such as increased VFA concentrations (particularly butyrate) and the hormonal effects of insulin and of insulin like growth factor 1 have partly been confirmed to be involved in the regulation of this adaptation process. More research is needed in order to determine the factors causally inducing adaptational responses and the amount of dietary change at which the adaptational responses apparently level off. Despite these uncertainties, an increase of energy and protein intake undoubtedly improves the barrier and transport functions of the epithelium. Consequently, at least for 2 weeks before parturition, a diet should be offered that is fermented more easily and more rapidly than the diet in the early dry period. Furthermore, efforts should be directed towards an improved voluntary food intake before and after parturition, with special attention being paid to the mechanisms enabling the epithelium to adapt to and handle the regular diurnal variations in pH, VFA concentration and osmotic pressure, mechanisms that seem to fail in the pathophysiology of subacute rumen acidosis.

Urea transport across the gastrointestinal tract involves transporters of the urea transporter-B group, the regulation of which is poorly understood. The classical stimulatory effect of CO2and the effect of short-chain fatty acids (SCFA) on the ruminal recycling of urea were investigated by using Ussing chamber and microelectrode techniques with isolated ruminal epithelium of sheep. The flux of urea was found to be phloretin sensitive and passive. At a luminal pH of 6.4, but not at 7.4, the addition of SCFA (40 mmol/l) or CO2/HCO3− (10% and 25 mmol/l) led to a fourfold increase in urea flux. The stepwise reduction of luminal pH in the presence of SCFA from 7.4 to 5.4 led to a bell-shaped modification of urea transport, with a maximum at pH 6.2. Lowering the pH in the absence of SCFA or CO2had no effect. Inhibition of Na+/H+exchange increased urea flux at pH 7.4, with a decrease being seen at pH 6.4. In experiments with double-barreled, pH-sensitive microelectrodes, we confirmed the presence of an apical pH microclimate and demonstrated the acidifying effects of SCFA on the underlying epithelium. We confirm that the permeability of the ruminal epithelium to urea involves a phloretin-sensitive pathway. We present clear evidence for the regulation of urea transport by strategies that alter intracellular pH, with permeability being highest after a moderate decrease. The well-known postprandial stimulation of urea transport to the rumen in vivo may involve acute pH-dependent effects of intraruminal SCFA and CO2on the function of existing urea transporters.

A previous study has shown that Cry1Ab, a lepidopteran-specific toxin derived from Bacillus thuringiensis, does not affect the vitality of cultured cells of the ruminal epithelium of the sheep. While this may be due to lack of specific receptors for toxin action, other mechanisms of resistance should also be considered. In order to directly assess the pore-forming potential of Cry1Ab, we studied the interaction of this toxin with isolated, perfused cells of the ruminal epithelium using the whole-cell and single-channel configurations of the patch-clamp technique. At concentrations found in vivo in the rumen of cows (10 ng/ml) and at a temperature of 37 degrees C, no significant effects of Cry1Ab could be observed. At 100 ng/ml, exposure of ruminal cells to Cry1Ab induced a significant rise in outward current in 16 of 34 cells, with a fourfold increase in the conductance for potassium. The cell membrane remained selective for potassium over sodium (p(K)/p(Na) = 1.8 + or - 0.3), with a considerable additional chloride conductance. In outside-out patches, exposure to high Cry1Ab concentrations induced channel-like events that reached levels of over 500 pS. We conclude that the unchanged vitality of intact ruminal epithelial cells exposed to Cry1Ab in vitro at high concentrations may be related to other factors besides the proposed absence of a specific receptor for the membrane insertion of this toxin.

Prostanoids and cannabinoids have ocular hypotensive and neuroprotective properties. The effect of the prostanoid AH13205 (EP2), the thromboxane-mimetic U46619, the cannabinoid (CB) agonists WIN55212-2 and CP 55,940, endothelin-1 (ET-1) and 8-bromo-cAMP on the membrane currents of trabecular meshwork (TM) cells were measured using the patch-clamp technique and compared to their effects on TM contractility. Previous studies show relaxation of TM to AH 13205 and other substances that elevate cAMP, while U46619 and endothelin-1 contract TM. This study shows that after contraction (100%) with carbachol (10−6 m ), the CB agonist CP 55,940 dose-dependently reduced contractility to 83±4% (n=9) (10−6 m ) and 61±10%, (n=7) (10−5 m ). In the presence of both the CB1 antagonist AM251 (10−6 m ) and CP 55,940 (10−5 m ), the contractile response to carbachol reached 84±3% (n=6) of the original level. In patch-clamp experiments, membrane permeable 8-bromo-cAMP (10−4 m ) had no effect on currents of TM cells. In contrast, AH 13205 and two cannabinoids reversibly enhanced outward current through high-conductance Ca2+-activated K+ channels (BKCa, BK, maxi-K) to the following values (in % of the initial value at 100 mV): AH 13205 (10−5 m ): 200±28% (n=6), CP 55,940 (10−6 m ): 196±33% (n=7), CP 55,940 (10−5 m ): 484±113% (n=7), WIN55212-2 (10−5 m ): 205±41% (n=10). Iberiotoxin (10−7 m ) completely blocked these responses. The current response to CP 55,940 (10−5 m ) could be partially blocked by the CB1 antagonist AM251 (10−6 m ). Conversely, the contractile agents in this study either caused a transient reduction in outward current (ET-1(5×10−8 m )) or had no effect (U46619 (10−6 m )). We conclude that stimulation of EP2 and CB1 receptors in TM is coupled to the activation of BKCa channels via a non-diffusible second messenger cascade. This effect may contribute to the relaxant activity of EP2 and CB1 agonists in isolated TM strips, modulating ocular outflow.

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na+ conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K+ permeability > Cs+ permeability > Na+ permeability. Conductance increased and rectification decreased when either Mg2+ or both Ca2+ and Mg2+ were removed from the internal or external solution or both. The conductance could be blocked by Ba2+, but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current ( Isc) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both Isc and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in Isc was followed by a subsequent increase. The cAMP-mediated increase in Isc was reduced by low serosal Na+ and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na+ reduced 28Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na+/Mg2+ exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.

Current models of aqueous humor outflow no longer treat trabecular meshwork (TM) as an inert tissue passively distended by the ciliary muscle (CM). Instead, ample evidence supports the theory that trabecular meshwork possess smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure. In this model, trabecular meshwork and ciliary muscle appear as functional antagonists, with ciliary muscle contraction leading to a distension of trabecular meshwork with subsequent reduction in outflow. and with trabecular meshwork contraction leading to the opposite effect. Smooth-muscle relaxing substances would therefore appear to be ideal candidates for glaucoma therapy with the dual goal of reducing intraocular pressure via the trabecular meshwork and of improving vascular perfusion of the optic nerve head. However, for such substances to effectively lower intraocular pressure, the effect on the ciliary muscle would have to he minimal. For this reason, more information is needed on the signalling processes involved in regulating trabecular meshwork and ciliary muscle contractility. This review attempts to outline current knowledge of signal transduction pathways leading to relaxation and contraction of ciliary muscle and trabecular meshwork. Pathways can be classified as involving or not involving changes of membrane voltage and of requiring or not requiring external calcium: possibly, other pathways exist. These different pathways involve different ion channels and isoforms of PKC and are expressed to a differing degree in ciliary muscle and trabecular meshwork, leading to differential responses when exposed to relaxing or contracting pharmacological agents. Some of these agents. like tyrosine kinase inhibitors and inhibitors of PKC. have been shown to relax trabecular meshwork while leaving ciliary muscle comparatively unaffected. This profile makes these substances appear as ideal drugs for simultaneously improving ocular outflow and retinal circulation, parameters that determine the time course of visual deterioration in glaucoma.

Trabecular f2 meshwork (TM), a smooth muscle-like tissue with contractile properties, is involved in the regulation of aqueous humor outflow. However, little is known about the regulation of Ca2+influx in trabecular meshwork cells. We investigated the influence of acetylcholine and tyrosine kinases on Ca2+conductances of bovine TM (BTM) and human TM (HTM) cells using the perforated-patch configuration of the patch-clamp technique and measurements of intracellular free Ca2+([Ca2+]i). Depolarization of the cells in the presence of 10 m m Ba2+or Ca2+led to an activation of inward currents at potentials positive to -30 mV with characteristics typical of L-type Ca2+currents: when using 10 m m Ba2+, maximal inward current and inactivation time constant (τ) increased; the L-type Ca2+channel blocker nifedipine (1 μ m ) reduced and the L-type Ca2+channel agonist BayK8644 (5 μ m ) enhanced maximal inward current. Acetylcholine (100 μ m ) and carbachol (1 μ m ) led to an increase in inward Ba2+current whereas application of the tyrosine kinase inhibitors genistein (50 μ m ) and lavendustin A (20 μ m ) resulted in a decrease in inward current. The application of daidzein (10 μ m ), an inactive analog of genistein had no effect. Depolarization of the cells with 135 m m K+or direct stimulation of L-type channels by application of BayK 8644 led to an increase in [Ca2+]i. Carbachol (1 μ m ) induced an increase in [Ca2+]iwhich was decreased by application of the tyrosine kinase inhibitor genistein (50 μ m ). We conclude that HTM and BTM cells express voltage-dependent L-type Ca2+channels that influence intracellular Ca2+concentration and thus may modulate TM contractility. The activity of L-type Ca2+currents is influenced by muscarinic agonists and tyrosine kinases.

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an ‘off’ signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.

The present study investigated the significance of apical transport proteins for ruminal acetate absorption and their interaction with different anions. In anion competition experiments in the washed reticulorumen, chloride disappearance rate (initial concentration, 28 mM) was inhibited by the presence of a short-chain fatty acid mixture (15 or 30 mM of each acetate, propionate, and butyrate). Disappearance rates of acetate and propionate, but not butyrate (initial concentration, 25 mM each) were diminished by 40 or 80 mM chloride. In isolated ovine ruminal epithelia mounted in Ussing chambers, an increase in chloride concentration from 4.5 to 90 mM led to a decrease of apical acetate uptake at a concentration of 0.5 mM. Mucosal nitrate inhibited acetate uptake most potently whereas sulfate had no effect. Decreasing mucosal pH from 7.4 to 6.1 approximately doubled uptake of acetate both at 0.5 and 10 mM, but this doubling was almost abolished when HCO3− was absent. The stimulated uptake at mucosal pH 6.1 consisted of a bicarbonate-dependent, nitrate-inhibitable part ( Km = 54 mM) and a bicarbonate-independent component ( Km = 12 mM) that was also sensitive to nitrate inhibition. Maximal uptake was three times larger for bicarbonate-dependent vs. bicarbonate-independent uptake. Mucosal addition of 200 μM DIDS, 400 μM p-chloromercuribenzene sulfonic acid, 800 μM p-hydroxymercuribenzoic acid, or 100 μM phloretin had no effects on acetate uptake although the latter two inhibited l-lactate uptake. Our data conclusively show a dominant involvement of proteins in apical acetate uptake. Previously described pH effects on acetate absorption originate mainly from modulation of acetate/bicarbonate exchange. Additionally, there is bicarbonate-independent uptake of acetate anions that is protein coupled but not via monocarboxylate cotransporter.

The transport of nitrogen across the rumen epithelium is characterized by absorption of ammonia from the rumen and by an influx of urea into the rumen. The transport rates of both compounds are large and exhibit wide variation. The transport of ammonia occurs in two forms: in the lipophilic form as NH3, the magnitude of which is linearly related to the pH in the ruminal fluid at pH values above 7, while at a physiological pH of 6.5 or lower, ammonia is predominantly absorbed as NH4+via putative potassium channels in the apical membrane. The uptake of NH4+depends on the potential difference of the apical membrane, Pda, and shows competition with K uptake. The pathway for basolateral exit of NH4+is unknown. Hence, the relative transport rates of NH3or NH4+are determined by the ruminal pH according to the Henderson–Hasselbalch equation. Transport of ammonia interacts with the transport of Na and Mg mainly via changes of the intracellular pH. Urea recycling into the rumen has been known for many years and the transport across the rumen epithelium is mediated via urea transporters in the luminal and basolateral membrane of the epithelium. Transport of urea occurs by simple diffusion, but is highly variable. A significant increase of urea influx is caused by the fermentation products CO2and short-chain fatty acids. Conversely, there is some evidence of inhibition of urea influx by ruminal ammonia. The underlying mechanisms of this modulation of urea transport are unknown, but of considerable nutritional importance, and future research should be directed to this aspect of ruminal transport.

Flufenamic acid relaxes trabecular meshwork, a smooth muscle-like tissue involved in the regulation of ocular outflow in the eye. In this study, we attempted to determine if ionic channels are involved in this response.Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique.In trabecular meshwork, flufenamic acid (10(-5) M) reversibly stimulated outward current to 406 +/- 71% of initial outward current level in BTM (n = 10) and 294 +/- 75% of initial current level in HTM (n = 12) in all cells investigated; no significant differences emerged. The response was dosage-dependent. Replacement of potassium in all solutions eliminated the response to flufenamic acid (n = 4, BTM). Blocking K(ATP ) channels with glibenclamide (10(-5) M, n = 6) and small-conductance calcium-activated potassium channels with apamin (10(-6) M, n = 5) had no effect. A direct effect on calcium channels could also not be detected. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by iberiotoxin (10(-7) M) suppressed 87 +/- 9% (n = 6; HTM) and 91 +/- 10% (n = 6; BTM) of the response. Depleting the cells of calcium did not significantly alter the response to flufenamic acid.Flufenamic acid stimulates maxi-K channels in trabecular meshwork of both human and bovine origin. This should lead to hyperpolarization, closure of L-type channels and lowered cytosolic calcium levels, possibly explaining the relaxation observed in response to this substance.

The aim of this study is to characterize the subtype of tyrosine kinase-regulated L-type Ca(2+) channels in retinal pigment epithelial (RPE) cells. Ca(2+) channel alpha1D-subunits were enriched by immunoprecipitation from membrane proteins isolated from rat RPE cells. Western blot analysis of the precipitates revealed coprecipitation of pp60(c-src). In addition, in precipitates obtained with antibodies against pp60(c-src), alpha1D-subunits were identified. The same was observed in immunoprecipitations from rat brain neurons. Tyrosine phosphorylation of alpha1D-subunits was confirmed using anti-phosphotyrosine antibodies. Ba(2+) currents through L-type channels in cultured rat RPE cells were increased by intracellular application of active pp60(c-src) (30 U/ml) (heat-inactivated pp60(c-src) had no effect). Thus, L-type channels of the neuroendocrine subtype can be expressed in epithelial cells and are activated by tyrosine kinase of the src subtype. This kind of regulation is also suggested for brain-derived neurons.

Ample evidence supports the theory that trabecular meshwork possesses smooth-muscle-like properties. Trabecular meshwork cells express a large number of transporters, channels and receptors, many of which are known to regulate smooth-muscle contractility. It has been shown that trabecular meshwork can be induced to contract and relax in response to pharmacological agents. In the model of the bovine eye, confirmed in some cases by experiments on primates, agents that contract trabecular meshwork reduce outflow. On the cellular level, this is coupled with depolarization and a rise in intracellular calcium. Relaxation of trabecular meshwork, on the other hand, appears to be coupled to a stimulation of the maxi-K channel, inducing hyperpolarization and a closure of L-type calcium channels. No significant differences between cells from a human and a bovine source emerged, either in classical measurements of membrane voltage, in measurements of intracellular calcium or patch-clamp experiments. Thus, pharmacological agents that relax trabecular meshwork seem promising candidates for further research – the ultimate goal being an improvement of glaucoma therapy in humans.

Publisher Summary This chapter presents evidence for the contractile properties of the trabecular meshwork (TM) and their effects on aqueous humor reabsorption. In a study discussed in the chapter, membrane voltage measurements and patch-clamp techniques were applied to cultured bovine and human TM and ciliary muscle (CM) cells. Measurements of isometric tension were performed on isolated TM and CM strips. Anterior segments of bovine eyes with well-preserved TM were perfused to measure the outflow rate. Cultured bovine and human TM cells showed voltage spikes typical of smooth muscle cells that were inhibited by nifedipine but insensitive to tetrodotoxin. The excitability of TM cells indicates that they function as contractile smooth muscle cells. There is no principal difference between human and bovine TM cells in terms of K + and Ca 2+ channels, functional receptors for endothelin, and the effects of cholinergic and adrenergic agonists. The concept of functional antagonism between TM and CM should be considered in the interpretation of the mechanism of action of currently used antiglaucoma drugs and for the search for effective new drugs.

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.