Chemical analysis of materials used in pig housing with respect to the safety of products of animal origin.

Bedding, environmental enrichment materials and disinfectant powders in pig farming are meant to ensure a hygienic bedding environment or allow pigs to perform explorative behaviour. To our knowledge, no legal regulation exists, that established maximum contents for undesirable substances, such as toxic metals, dioxins or trace elements in these materials, although oral ingestion could be expected. In the present study, a total of 74 materials (disinfectant powders [n = 51], earth/peat [n = 12], biochar [n = 8], recycled manure solids [n = 3]) were analysed for their content of various toxic metals, trace elements, dioxins and polychlorinated biphenyls. The data suggest that, in some samples, trace elements like iron, copper and zinc might have been added intentionally in order to induce physiological effects (iron supply to piglets, copper and zinc as growth promoter in pigs). Moreover, some materials contained high levels of lead, cadmium or arsenic. Consequently, if farm animals repeatedly consume environmental enrichment and bedding materials or disinfectant powders in considerable amounts and these quantities are added to the daily ration, the amount of ingested undesirable substances and trace elements might exceed the maximum levels set for complete feedstuffs, and an elevated transfer into food of animal origin might occur. Future studies are required to address the possible quantitative contribution in the light of feed and food safety. Finally, the excretion of undesirable substances with manure needs to be considered due to their possible accumulation in soils.

Perfluoroalkyl substances (PFAS) in beaked redfish (Sebastes mentella) and cod (Gadus morhua) from arctic fishing grounds of Svalbard.

The present study gives an overview about the concentration of PFAS in liver, fillet and belly flap of beaked redfish (Sebastes mentella) and cod (Gadus morhua) caught in pristine arctic fishing grounds of Svalbard. Out of 17 analysed substances, only six perfluoroalkyl acids (PFAAs) could be detected in the fish. The most frequently quantified substances were PFOS and perfluoroundecanoic acid (PFUnA) in liver (100%) and fillet (at least 40% and 70%, respectively) of beaked redfish and cod, and in belly flap of beaked redfish (100%). Compared to cod, beaked redfish showed significant higher PFAA concentrations with highest levels in liver. Multiple comparisons of group differences for PFAA concentrations among fish species and matrices were independent of the evaluation method, but not for the PFAA-pattern analysis. The risk assessment of PFOS indicated that beaked redfish and cod caught in the Barents Sea can be a relevant exposure source for consumers.

Suitability of Wild Boar (Sus scrofa) as a Bioindicator for Environmental Pollution with Perfluorooctanoic Acid (PFOA) and Perfluorooctanesulfonic Acid (PFOS).

Wildlife species, such as roe deer, moose, brown hare, wild boar, etc., are known to accumulate persistent environmental contaminants and thus are useful as bioindicators for environmental pollution. Wild boars become exposed to perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) from flora, fauna, water, and soil. The main exposure pathway to PFOA and PFOS is assumed to be the oral intake. From studies in domestic pigs (belonging to the same species Sus scrofa), it has been established that the oral exposure results in the liver accumulation of PFOA and PFOS. Thus, we posit that wild boars can be quantitatively used as suitable bioindicators for the presence of these substances in the environment. After the environmental pollution case in the Hessian region Sauerland in 2006, monitoring programs of individual Federal States from 2007 to 2013 showed that almost all wild boar liver samples contained PFOA and PFOS. In 2014, the analyses of PFOA and PFOS in liver of wild boars hunted in the south, north, and west of Germany showed liver concentrations at the same level among regions. Overall, an average ratio of PFOS:PFOA concentration in liver of 20.5:1 was found. To estimate the actual ratio of PFOS:PFOA in the wild boars' dietary exposure, we performed toxicokinetic modeling. According to the model, the PFOS exposure is only 2.2 times that of PFOA (because PFOS has slower elimination kinetics and higher affinity for the liver than PFOA). Overall, the determination of PFOA and PFOS in liver of wild boars indicates that both substances are ubiquitously distributed in the environment. At the same time, higher exposures were found for animals living in closer proximity to dense human populations.

Investigations on the possible impact of a glyphosate-containing herbicide on ruminal metabolism and bacteria in vitro by means of the 'Rumen Simulation Technique'.

AIMS: This study was performed in a well-established in vitro model to investigate whether the application of a glyphosate-containing herbicide might affect the bacterial communities and some biochemical parameters in a cow's rumen. METHODS AND RESULTS: The test item was applied in two concentrations (high and low) for 5 days. In a second trial, fermentation vessels were inoculated with Clostridium sporogenes before the high dose was applied. Effluents were analysed by biochemical, microbiological and genetic methods. A marginal increase in short-chain fatty acid production and a reduction in NH3 -N were observed. There were minor and rather equivocal changes in the composition of ruminal bacteria but no indications of a shift towards a more frequent abundance of pathogenic Clostridia species. Clostridium sporogenes counts declined consistently. CONCLUSIONS: No adverse effects of the herbicide on ruminal metabolism or composition of the bacterial communities could be detected. In particular, there was no evidence of a suspected stimulation of Clostridia growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic activity of glyphosate resulting in microbial imbalances has been postulated. In this exploratory study, however, intraruminal application of concentrations reflecting potential exposure of dairy cows or beef cattle did not exhibit significant effects on bacterial communities in a complex in vitro system. The low number of replicates (n = 3/dose) may leave some uncertainty.

Excretion pathways and ruminal disappearance of glyphosate and its degradation product aminomethylphosphonic acid in dairy cows.

From 6 balance experiments with total collection of feces and urine, samples were obtained to investigate the excretion pathways of glyphosate (GLY) in lactating dairy cows. Each experiment lasted for 26d. The first 21d served for adaptation to the diet, and during the remaining 5d collection of total feces and urine was conducted. Dry matter intake and milk yield were recorded daily and milk and feed samples were taken during the sampling periods. In 2 of the 6 experiments, at the sampling period for feces and urine, duodenal contents were collected for 5d. Cows were equipped with cannulas at the dorsal sac of the rumen and the proximal duodenum. Duodenal contents were collected every 2h over 5 consecutive days. The daily duodenal dry matter flow was measured by using chromium oxide as a volume marker. All samples (feed, feces, urine, milk and duodenal contents were analyzed for GLY and aminomethylphosphonic acid (AMPA). Overall, across the 6 experiments (n=32) the range of GLY intake was 0.08 to 6.67mg/d. The main proportion (61±11%; ±SD) of consumed GLY was excreted with feces; whereas excretion by urine was 8±3% of GLY intake. Elimination via milk was negligible. The GLY concentrations above the limit of quantification were not detected in any of the milk samples. A potential ruminal degradation of GLY to AMPA was derived from daily duodenal GLY flow. The apparent ruminal disappearance of GLY intake was 36 and 6%. In conclusion, the results of the present study indicate that the gastrointestinal absorption of GLY is of minor importance and fecal excretion represents the major excretion pathway. A degradation of GLY to AMPA by rumen microbes or a possible retention in the body has to be taken into account.

Can perfluoroalkyl acids biodegrade in the rumen simulation technique (RUSITEC)?

BACKGROUND: The behaviour of perfluoroalkyl acids (PFAAs) in tissues of ruminants has been shown to differ from that of monogastrics (J Agric Food Chem 61(12):2903-2912 doi:10.1021/jf304680j, 2013; J Agric Food Chem 62(28):6861-6870, 2014). This may be a consequence of the complex microbial ecosystem in the rumen. To evaluate this hypothesis, the recovery of PFAAs was studied using the rumen simulation technique as an indication for biodegradation in rumen. The PFAA-recovery from a microbial fermentation of feed containing PFAAs was compared to the same feed in the absence of ruminal microorganisms (MOs). RESULTS: Release of PFAAs from feed into fermentation fluid was found to be faster for perfluorobutane sulfonic acid (PFBS) than for perfluorooctane sulfonic acid (PFOS). Differences between perfluoroalkyl carboxylic acids (PFCAs) could not be observed. Proportions of PFAAs recovered in the fermentation fluids decreased by increasing chain lengths for the perfluoroalkyl sulfonic acids (PFSAs) (31 % PFBS, 28 % perfluorohexane sulfonic acid [PFHxS], 20 % perfluoroheptane sulfonic acid [PFHpS], 11 % PFOS) and PFCAs (33 % perfluorohexane carboxylic acid [PFHxA], 32 % perfluoroheptane carboxylic acid [PFHpA], 24 % perfluorooctanoic acid [PFOA]). In contrast, levels in feed increased with increasing chain length for both PFSAs and PFCAs. CONCLUSION: The attachment of MOs to feed particles was assumed to account for higher PFAA levels in fermented feeds and for lower levels in the fermentation fluids. Total recovery of PFAAs was significantly lower in presence of ruminal MOs compared to experimental procedure under sterile conditions. Although, there are optimal reductive conditions for MOs in rumen, our results do not univocally indicate whether PFAAs were degraded by ruminal fermentation.

Transfer of perfluorooctane sulfonic acid (PFOS) from contaminated feed to dairy milk.

Dietary intake is the predominant route for human exposure to perfluorooctane sulfonic acid (PFOS). Single pollution events may thus affect human exposure if polluted ground and water is used to produce animal feed or food. In this study, a physiologically based pharmacokinetic (PBPK-) model is derived that describes the uptake of PFOS from contaminated feed by cows and its subsequent elimination through the cows' milk. Parameter values of the model were estimated by fitting to experimental data of a cow feeding trial. Model calculations showed that almost all PFOS ingested is excreted through the cows' milk. The elimination rate, however, was low as the estimated half-life in the cow was 56days and it may, thus, take a long time after an initial pollution event to produce PFOS-free milk. The derived model can be used to estimate the transfer of PFOS through the dairy food chain and can be used for comparison of various contamination routes.

The role of dietary selenium in bovine mammary gland health and immune function.

Mastitis is not only a major cause of economic losses to the dairy industry but also a major problem in ensuring the quality and safety of the milk, associated with high somatic cell counts and residues of antibiotics used for treatment. One innovative approach to protection against mastitis is to stimulate the animal's natural defense mechanisms. Technological advances in immunological research have increased our ability to exploit the immunity of the bovine mammary gland during periods of high susceptibility to disease. The trace element selenium affects the innate and the adaptive immune responses of the mammary gland through cellular and humoral activities. Substantial research has been carried out on the effect of selenium (Se) on the immune function of the mammary gland and subsequent improvement in bovine udder health and mastitis control. Levels higher than current recommendations and Se-yeast can potentially be used to enhance our capacity to modulate the physiological mechanisms of the bovine mammary gland to respond to infection. This article provides an overview of the most recent research in this field.

Mechanisms of action of unoprostone on trabecular meshwork contractility.

PURPOSE: This study was performed to clarify the possible mechanism behind the ocular hypotensive effect of unoprostone isopropyl (Rescula; Novartis Ophthalmics AG, Basel, Switzerland), a new docosanoid that has been shown to reduce intraocular pressure (IOP) in patients with ocular hypertension or primary open-angle glaucoma. To gain insight into the possible mode of action, the effects of unoprostone on ciliary muscle (CM) and trabecular meshwork (TM) contractility, intracellular calcium levels, and membrane channels were investigated. METHODS: The effects of unoprostone (M1 metabolite = free acid, 10(-5) M) and endothelin (ET)-1 (10(-9) M) on bovine TM (BTM) and ciliary muscle (CM) strips were investigated, by using a custom-made force-length transducer system. The effects of unoprostone and ET-1 (5 x 10(-8) M) on intracellular Ca(2+) mobilization in cultured human TM (HTM) were measured using fura-2AM as a fluorescent probe. Patch-clamp experiments were performed on HTM and BTM cells to investigate the unoprostone-dependent modulation of membrane currents. RESULTS: In isolated TM and CM strips, unoprostone almost completely inhibited ET-induced contractions (TM: 2.9% +/- 4.3% vs. 19.6% +/- 5.7%, P < 0.05, n = 6; CM: 1.4% +/- 1.6% vs. 30.1% +/- 5.3%, P < 0.01, n = 6; 100% = maximal carbachol-induced (10(-6) M) contraction). However, neither carbachol-induced contraction nor baseline tension was affected by unoprostone. Furthermore, unoprostone had no effect on baseline intracellular calcium levels (baseline: 126 +/- 45 nM versus unoprostone: 132 +/- 42 nM, n = 8) in HTM cells. The endothelin-induced increase (679 +/- 102 nM), however, was almost completely (P < 0.01) blocked by unoprostone (178 +/- 40 nM). In patch-clamp recordings, unoprostone could be shown to double the amplitude of outward current (HTM: 200% +/- 33%; n = 6; BTM: 179% +/- 20%; n = 8). This effect was blocked by the specific inhibitor of maxi-K channels, iberiotoxin. CONCLUSIONS: This study presents evidence for direct interaction of unoprostone with the contractility of the TM and CM. This compound may lower IOP by affecting aqueous outflow, most probably conventional outflow pathways (i.e., TM) through inhibition of ET-dependent mechanisms. In addition, unoprostone interacts with the maxi-K channel. Although primarily Ca(2+)-sensitive signal-transduction pathways seem to be involved, effects of unoprostone on Ca(2+)-independent pathways and uveoscleral outflow cannot be excluded.

Flufenamic acid enhances current through maxi-K channels in the trabecular meshwork of the eye.

PURPOSE: 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. METHODS: Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique. RESULTS: 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. CONCLUSIONS: 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.

Muscarinic receptors of the M2 subtype in human and bovine trabecular meshwork.

BACKGROUND: The trabecular meshwork is a tissue actively involved in the regulation of intraocular pressure via contractile mechanisms. The present study was performed to investigate the effects of muscarinic m2-receptor antagonists on trabecular meshwork contractility and to identify the m2 muscarinic receptor in human and bovine trabecular meshwork cells. METHODS: Isometric tension measurements of bovine trabecular meshwork strips were performed using a custom-made force length transducer. Western blot and immunoprecipitation analysis was used to detect the m2-receptor proteins in membrane preparations of human and bovine trabecular meshwork cells. RESULTS: Immunoblotting results showed the expression of an m2-receptor protein band at 56 kDa in both human and bovine trabecular meshwork cells. Two different m2-receptor antagonists were tested on trabecular meshwork contractility. After carbachol-induced contraction (10(-6) M set to 100% contractile force), specific m2-receptor antagonists were applied. 3 alpha-Chloroimperaline (10(-6) M) had no effect on the maximal carbachol-induced contraction in trabecular meshwork strips. Methoctramine induced a significant relaxation at concentrations of 10(-7), 10(-6) and 5 x 10(-6) M even in the presence of m1- and m3-receptor antagonists. CONCLUSION: These data indicate that in addition to the m3-receptor subtype present in the trabecular meshwork this tissue also features the m2 receptor. This receptor is partly involved in the regulation of trabecular meshwork contractility, suggesting that outflow facility might be influenced through this receptor.

Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A.

PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells. CONCLUSIONS: The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.

The regulation of trabecular meshwork and ciliary muscle contractility.

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.

Influence of muscarinic agonists and tyrosine kinase inhibitors on L-type Ca(2+)Channels in human and bovine trabecular meshwork cells.

Trabecularmeshwork (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 Ca(2+)influx in trabecular meshwork cells. We investigated the influence of acetylcholine and tyrosine kinases on Ca(2+)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 Ca(2+)([Ca(2+)](i)). Depolarization of the cells in the presence of 10 m m Ba(2+)or Ca(2+)led to an activation of inward currents at potentials positive to -30 mV with characteristics typical of L-type Ca(2+)currents: when using 10 m m Ba(2+), maximal inward current and inactivation time constant (tau) increased; the L-type Ca(2+)channel blocker nifedipine (1 microm) reduced and the L-type Ca(2+)channel agonist BayK8644 (5 microm) enhanced maximal inward current. Acetylcholine (100 microm) and carbachol (1 microm) led to an increase in inward Ba(2+)current whereas application of the tyrosine kinase inhibitors genistein (50 microm) and lavendustin A (20 microm) resulted in a decrease in inward current. The application of daidzein (10 microm), 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 [Ca(2+)](i). Carbachol (1 microm) induced an increase in [Ca(2+)](i)which was decreased by application of the tyrosine kinase inhibitor genistein (50 microm). We conclude that HTM and BTM cells express voltage-dependent L-type Ca(2+)channels that influence intracellular Ca(2+)concentration and thus may modulate TM contractility. The activity of L-type Ca(2+)currents is influenced by muscarinic agonists and tyrosine kinases.

Regulation of trabecular meshwork contractility.

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.

The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility.

PURPOSE: The possible role of protein kinase C (PKC) inhibitors in novel pressure-lowering drugs is currently under investigation. To gain further insight into regulation of contractility by PKC in trabecular meshwork (TM) and ciliary muscle (CM), the effects of various PKC inhibitors and activators were tested. METHODS: Isometric tension measurements of bovine TM and CM strips were performed. PKC was stimulated by phorbol ester and by the diacylglycerol analogue diC8. PKC blockade was accomplished using H7 and myristoilated PKC substrate (mPKC). Western blot analysis was used to identify specific PKC isoforms in human trabecular meshwork (HTM), human ciliary muscle (HCM), and bovine TM and CM. RESULTS: In tissues precontracted by carbachol PKC antagonist H7 led to a relaxation of TM (25+/-7.2 versus 100%; n = 8) with no effect on CM. mPKC substrate selectively blocks PKC. This substance led to relaxation of TM (32.8+/-7.4 versus 100%, n = 7), whereas CM was not affected. PMA at concentrations of 10(-6) M led to a slow contraction of both tissues that was more marked in TM. DiC8 and 4alpha-phorbol had no effect on contractility. Western blot analysis revealed expression of calcium-dependent PKC-alpha and calcium-independent PKC-epsilon isoforms in HTM and HCM. PKC-epsilon expression was more pronounced in HTM than in HCM. Similar PKC isoform expression was found in native bovine tissue. CONCLUSIONS: PKC isoforms show different tissue distributions in human and bovine TM and CM. Contractility differences exist in both tissues in response to PKC antagonists and agonists. The data indicate that PKC may be involved in regulation of aqueous humor outflow by the TM. Thus, inhibition of PKC may represent a new way of influencing outflow facility through isolated relaxation of TM.

Stimulation of maxi-K channels in trabecular meshwork by tyrosine kinase inhibitors.

PURPOSE: Muscarinic agonists contract and tyrosine kinase inhibitors relax precontracted trabecular meshwork, a smooth muscle-like tissue involved in the regulation of aqueous humor outflow. The effect of tyrosine kinase inhibitors on membrane currents of cells stimulated by acetylcholine was examined. METHODS: Cells from bovine trabecular meshwork were studied using both the perforated patch-clamp technique with nystatin and the single-channel technique. RESULTS: Application of the tyrosine kinase inhibitor genistein (5 x 10(-5) M) on trabecular meshwork cells stimulated with acetylcholine resulted in a reversible increase in outward current to 578%+/-154% (n = 16) of the initial current level. The effect of genistein was dose dependent. Reversal potential was hyperpolarized by 15+/-3 mV (n = 9). Tyrphostin 51, a synthetic inhibitor of tyrosine kinases, had the same effect (433%+/-46%; n = 7). Daidzein, a nonactive structural analogue of genistein, had no effect (n = 4). The stimulation of outward current by tyrosine kinase inhibitors was blocked by substitution of tetraethylammonium (TEA+) for potassium, whereas the potassium channel blockers glibenclamide (K-ATP) and apamin (low-conductance calcium-activated potassium channel) had no effect. Blockage of the high-conductance calcium-activated potassium channel (maxi-K) by charybdotoxin or iberiotoxin (10(7) M) suppressed 86%+/-18% (n = 4) of the response. Depleting the cells of calcium did not have an effect on the current stimulated by genistein. In the excised inside-out configuration, open probability increased to 417%+/-39% (n = 3) after exposure to genistein. CONCLUSIONS: In trabecular meshwork, tyrosine kinase inhibitors activate maxi-K (K(Ca)) channels. Hyperpolarization caused by efflux of potassium could lead to the relaxation of trabecular meshwork by tyrosine kinase inhibitors.

Involvement of protein tyrosine kinase in the InsP3-induced activation of Ca2+-dependent Cl- currents in cultured cells of the rat retinal pigment epithelium.

This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]i) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl- channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 microM) led to an increase in [Ca2+]i and activation of Cl- currents. In contrast, intracellular application of Ca2+ (10 microM) only induced transient activation of Cl- currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of ICRAC, La3+ (10 microM), despite the fact that both maneuvers led to a decline in [Ca2+]i. The InsP3-induced rise in Cl- conductance could be prevented either by thapsigargin-induced (1 microM) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 microM) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 microM) reduced Cl--conductance in 50% of the cells investigated without affecting [Ca2+]i. Inhibition of protein tyrosine kinase (50 microM tyrphostin 51, 5 microM genistein, 5 microM lavendustin) reduced an increase in [Ca2+]i and Cl- conductance. In summary, elevation of [Ca]i by InsP3 leads to activation of Cl- channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.

The Royal College of Surgeons rat: an animal model for inherited retinal degeneration with a still unknown genetic defect.

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.