Recovery of human motoneurons during rotation.

During prolonged contractions, few studies have reported rotation among low threshold motoneurons. The question arises whether a motoneuron stops firing due to an increase in firing threshold or whether it is due to regional switching of activity among muscle fascicles. We postulated that if the rest period resulted from an increase in firing threshold, a progressive recovery in the excitability of the motoneuron would be observed during the rest period. The excitability of soleus or tibialis anterior motoneurons was tested during the rest periods. The results showed that a previously tonic motoneuron that had dropped off during rotation, rarely responded to Ia or TMS inputs in the initial parts of the rest period; however, its response probability increased significantly in the second half. Based on these data, we suggest that the observed rotation is due to changes in firing thresholds of motoneurons during prolonged firing.

Influence of supplemental dietary poultry fat on the digestive and reproductive organ characteristics of commercial layers inoculated before or at the onset of lay with F-strain Mycoplasma gallisepticum.

Effects of F-strain Mycoplasma gallisepticum (FMG) inoculation and 1.5% supplemental dietary poultry fat (PF) on the digestive and reproductive organ characteristics of commercial layers at 58 wk of age were investigated. Sham and FMG inoculations were administered at 12 (before lay) and 22 (early in lay) wk of age, and dietary treatments (basal control diets and basal control diets with PF) were initiated at 20 wk of age. Supplemental PF increased BW and decreased isthmal length relative to total oviduct length in hens. Various oviduct segments were also affected by the type and age of inoculation, and these effects were further influenced by the use of PF. In comparison to their time-specific sham-inoculated controls, infundibulum weight relative to BW was increased when birds were inoculated with FMG at 22 wk, whereas isthmus weight relative to total oviduct weight was increased by FMG inoculation at 12 wk of age. However, PF affected infundibulum length relative to total oviduct length only in sham-inoculated birds, and PF increased magnum weight relative to total oviduct weight only in birds inoculated at 22 wk of age (sham or FMG). Furthermore, PF decreased isthmus weight relative to total oviduct weight only in birds that were sham-inoculated (12 or 22 wk). In conclusion, the inoculation of FMG at 12 or 22 wk may increase the relative contributions of the isthmus and infundibulum, respectively, to the total mass of the oviduct. In addition, PF may decrease the relative length of the isthmus and increase the relative weight of the magnum in the oviducts of birds that have been inoculated at 22 wk of age (sham or FMG). Previous studies have shown 1.5% supplemental dietary PF to influence feed consumption throughout lay and performance early in lay in hens that were inoculated with FMG at 12 wk of age. However, the current results suggest that these influences are associated with gross changes in the oviduct but not the digestive tract of layers.

Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.

Comprehensive genomic analysis of the important human pathogen Staphylococcus aureus was achieved by a strategy involving antisense technology in a regulatable gene expression system. In addition to known essential genes, many genes of unknown or poorly defined biological function were identified. This methodology allowed gene function to be characterized in a comprehensive, defined set of conditionally growth-defective/lethal isogenic strains. Quantitative titration of the conditional growth effect was performed either in bacterial culture or in an animal model of infection. This genomic strategy offers an approach to the identification of staphylococcal gene products that could serve as targets for antibiotic discovery.

Influence of supplemental dietary poultry fat on the performance of commercial layers inoculated before or at the onset of lay with F-strain Mycoplasma gallisepticum.

The effects of F-strain Mycoplasma gallisepticum (FMG) inoculation and 1.5% supplemental dietary poultry fat (PF) on the performance of commercial layers between 20 and 58 wk of age were investigated. Sham and FMG inoculations were administered at 12 (before lay) and 22 (early in lay) wk and dietary treatments (basal control diets and basal control diets with 1.5% supplemental PF) were initiated at 20 wk of age. Mortality at wk 47 and 53 was greatest in birds inoculated with FMG at 22 wk. Feed consumption from 20 to 23 and from 52 to 55 wk of age was greater in birds that were inoculated with FMG (12 or 22 wk). However, feed consumption decreased in birds that were inoculated at 12 wk (sham or FMG) when 1.5% supplemental PF was added to the diet. Percentage of total egg production (EP) between 22 and 58 wk of age was highest in hens that were inoculated with FMG at 22 wk. Furthermore, weekly EP increased at wk 27 and 58 and decreased at wk 47 after birds had been inoculated with FMG (12 or 22 wk), and increased at 22 wk and decreased at 54 wk when inoculations (sham or FMG) were given at 22 wk. Egg weight was increased at wk 29, 31, 39, 40, 42, 44, 53, and 58 in birds that were inoculated with FMG (12 or 22 wk); however, there were no coherent treatment effects on eggshell quality. An FMG inoculation at 22 wk may promote total EP through 58 wk, whereas the inoculation of commercial layers with FMG (12 or 22 wk) may increase subsequent feed consumption during the early and late stages of EP and increase egg weight throughout lay. However, the supplementation of hen diets with 1.5% PF beginning at 20 wk of age may reduce subsequent feed consumption throughout lay in birds having experienced a prelay (12 wk) inoculation.

Influence of supplemental dietary poultry fat on the yolk characteristics of commercial layers inoculated before or at the onset of lay with F-strain Mycoplasma gallisepticum.

Effects of F-strain Mycoplasma gallisepticum (FMG) inoculation and 1.5% supplemental dietary poultry fat (PF) on the egg yolk characteristics of commercial layers at 24, 34, 44, 50, and 58 wk of age were investigated. Sham and FMG inoculations were administered at 12 and 22 wk of age and dietary treatments (basal control and basal control with 1.5% supplemental PF) were initiated at 20 wk of age. Yolk lipid concentration was reduced on wk 24 in birds that had been inoculated at 12 or 22 wk of age with FMG. The use of 1.5% supplemental PF increased percentage of yolk weight and yolk:albumen ratio across age and inoculation treatment. At 58 wk of age, concentrations of yolk palmitic acid increased and those of oleic and linolenic acid decreased when sham inoculations were given at 22 rather than at 12 wk of age. However, FMG inoculations given at 22 rather than at 12 wk increased palmitoleic acid and decreased stearic acid yolk concentrations. At 12 wk of age, FMG inoculations decreased yolk palmitoleic, oleic, and linolenic acid concentrations while causing increased yolk stearic and arachidonic acid levels when compared with sham inoculations. Furthermore, 1.5% supplemental PF decreased concentrations of palmitic and oleic acid and increased those of linoleic acid in the yolk at 58 wk of age. Despite the interaction of 1.5% supplemental PF with the prelay inoculation of FMG on early (18 to 26 wk) layer performance noted in a previous report, the effects of a prelay FMG inoculation and 1.5% supplemental PF on the egg yolk characteristics examined in the current study were independent of each other. This suggests that 1.5% supplemental PF is not effective in modulating the effects of an FMG inoculation at 12 wk of age on hen egg yolk characteristics between 24 and 58 wk of age and that the combined effects of PF supplementation and FMG inoculation on performance do not influence egg yolk characteristics.

Effects of supplemental dietary phytase and 25-hydroxycholecalciferol on the performance characteristics of commercial layers inoculated before or at the onset of lay with the F-strain of Mycoplasma gallisepticum.

The effects of dietary supplementation with phytase and 25-hydroxycholecalciferol on the performance characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum were assessed. Experimental layer diets, which included a basal control diet or the same diet supplemented with 0.025% phytase and 25-hydroxycholecalciferol, were fed from 20 through 58 wk of age. Weekly and total egg production were determined from 22 through 58 wk, and egg weight and various internal egg and eggshell quality characteristics were examined at 34, 50, and 58 wk of age. F-strain M. gallisepticum inoculation decreased egg production at the beginning of lay (wk 22 and 23) but increased post-peak lay at wk 45. However, there were no treatment effects of any kind on total egg production, egg weight, or any of the internal egg and eggshell characteristics examined during lay. In conclusion, dietary supplementation with phytase and 25-hydroxycholecalciferol did not affect layer performance or interact with the effects of F-strain M. gallisepticum inoculation; however, F-strain M. gallisepticum inoculation resulted in a shift in egg production from wk 22 to 45 without having an overall effect on total egg production.

Evaluation of an experimental chlorate product as a preslaughter feed supplement to reduce salmonella in meat-producing birds.

A study was conducted to evaluate the effect of experimental chlorate product (ECP) feed supplementation on Salmonella Typhimurium (ST) in the crop and ceca of market-age broilers. In trial 1, 160 market-age broilers were randomly assigned to 8 treatment groups and replicated twice, with 20 broilers per pen for 1 wk. Trial 2 used the same design, but used 80 market-age broilers with 10 broilers per pen. Treatments were as follows: 1) control feed + double-distilled drinking water (dd H(2)O); 2) control + 18.5% experimental zeolite carrier with dd H(2)O; 3 to 7) control feed supplemented with 0.5, 1.0, 5.0, 10.0, or 18.5% of a feed grade ECP + dd H(2)O; 8) control feed + 1x ECP (0.16% w/v; containing 15 mM chlorate ion equivalent) added to dd H(2)O. Seven-week-old broilers were provided experimental treatments for 7 d, killed, and then ceca and crops were removed and evaluated for ST. Broilers fed 5 to 18.5% ECP or water ECP had a significantly lower (P < 0.05) incidence of ST in the crop (36 to 38% and 14%, respectively) when compared with the control (60%). Broilers fed 10% ECP or water ECP had significantly lower ST crop concentrations (1.03 log(10) and 0.38 log(10) ST/g, respectively) when compared with broilers fed a control diet (1.54 log(10) ST/g). Crop and ceca ST incidence (32 to 48%) and concentration (1.00 to 1.82 log(10) ST/g) were significantly lower in broilers fed 5 to 18.5% ECP as compared with the control (78%; 2.84 log(10) ST/g). Broilers fed 5% or greater ECP had significantly higher water consumption (380 to 580 mL water/d) and litter moisture (31 to 56%) when compared with the control (370 mL water/d; 23% moisture). Only broilers fed 18.5% ECP had significantly lower 7-wk BW (2.77 kg of BW) when compared with the controls (3.09 kg of BW). Average daily gains were significantly depressed in broilers fed 10 or 18.5% ECP compared with the controls. These results indicate broilers supplemented with feed

Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains.

BACKGROUND AND PURPOSE: The small and intermediate conductance, Ca2+-sensitive K+ channels (SK(Ca) and IK(Ca), respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK(Ca) and SK(Ca) channels was investigated. EXPERIMENTAL APPROACH: The effect of the caveolae-disrupting agent methyl-beta-cyclodextrin (MbetaCD) on IK(Ca) and SK(Ca) localization and function was determined. KEY RESULTS: EDHF-mediated, SK(Ca)-dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK(Ca) with TRAM-34) were inhibited by MbetaCD. Hyperpolarizations evoked by direct SK(Ca) channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbetaCD, an effect reversed by cholesterol. In contrast, IK(Ca)-dependent hyperpolarizations (in the presence of apamin) were unaffected by MbetaCD. Similarly, in porcine coronary arteries, EDHF-mediated, SK(Ca)-dependent (but not IK(Ca)-dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbetaCD. In mesenteric artery homogenates subjected to sucrose-density centrifugation, caveolin-1 and SK3 (SK(Ca)) proteins but not IK1 (IK(Ca)) protein migrated to the buoyant, caveolin-rich fraction. MbetaCD pretreatment redistributed caveolin-1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin-1 were co-localized. Furthermore, caveolin-1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein. CONCLUSIONS AND IMPLICATIONS: These data provide strong evidence that endothelial cell SK(Ca) channels are located in caveolae while the IK(Ca) channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.

Regulation of c-SRC activity and function by the adapter protein CAS.

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.

Effects of supplemental dietary phytase and 25-hydroxycholecalciferol on the digestive and reproductive organ characteristics of commercial layers inoculated before or at the onset of lay with the F-strain of Mycoplasma gallisepticum.

In 3 trials, the effects of dietary supplementation with phytase (PHY) and 25-hydroxycholecalciferol (25-D3) on the digestive and reproductive organ characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum (FMG) were assessed at 58 wk of age. Experimental layer diets that included a basal control diet or a control diet supplemented with 0.025% PHY and 25-D3 were fed from 20 through 58 wk of age. As a percentage of total oviduct weight, magnum weight was lower in birds that were inoculated (sham or FMG) at lay onset compared with those that were inoculated prelay, and in FMG-inoculated birds, relative duodenum length was greater in those inoculated at 12 compared with 22 wk. Also, as percentages of organ weight or length, infundibulum length and isthmus weight were increased, whereas duodenum length was decreased by dietary supplementation with PHY and 25-D3. The overall timing (12 vs. 22 wk) of inoculation can affect the reproductive organ characteristics of layers, whereas, more specifically, the timing of an FMG inoculation may affect their digestive organ structure. Furthermore, independent of inoculation timing and type, the reproductive organ and digestive systems of laying hens may be influenced by dietary supplementation with PHY and 25-D3.

Effects of supplemental dietary phytase and 25-hydroxycholecalciferol on the blood characteristics of commercial layers inoculated before or at the onset of lay with the F-strain of Mycoplasma gallisepticum.

In 3 trials, the effects of dietary supplementation with phytase (PHY) and 25-hydroxycholecalciferol on BW and the blood characteristics of commercial layers that were inoculated prelay (12 wk of age) or at the onset of lay (22 wk of age) with F-strain Mycoplasma gallisepticum were assessed at 34, 50, and 58 wk of age. Experimental layer diets, which included either a basal control diet or the same diet supplemented with 0.025% PHY and 25-hydroxycholecalciferol, were fed from 20 through 58 wk of age. The supplemented diet decreased blood hematocrit values across bird age, inoculation type (sham vs. F-strain M. gallisepticum), and age of inoculation (prelay vs. onset of lay). Phytase- and 25-hydroxycholecalciferol-supplemented diets reduced bird BW in sham-inoculated control birds across bird age and age of inoculation. This effect was not observed in F-strain M. gallisepticum-inoculated birds. Furthermore, across diet (control vs. supplemented) and inoculation type, total plasma protein concentration at 34 wk of age was higher in birds that were inoculated at the onset of lay compared with those inoculated prelay. Diet, inoculation type, and inoculation age had no effect on mortality, reproductive organ histopathological lesion scores, or serum cholesterol and Ca concentrations. In conclusion, throughout lay, the supplementation of commercial layer diets with PHY may lower hematocrit, and inoculation with F-strain M. gallisepticum prelay or at the onset of lay may ameliorate the depressing effects of dietary PHY and 25-hydroxycholecalciferol supplementation on hen BW.

Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition.

The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.

The identification of p130cas-binding proteins and their role in cellular transformation.

Adaptor proteins play an important role in signal transduction by regulating the establishment and maintenance of functionally important protein complexes. A recently described member of this group of proteins is p130cas (CAS), which contains numerous sequence motifs predicted to be involved in mediating protein-protein interactions. We propose that adaptor molecules like CAS may help determine the response of a cell to a particular signal by interacting with specific subsets of cellular proteins. To test this hypothesis, we have identified potential binding partners of CAS that may play a rote in cellular transformation by the oncoproteins v-SRC and/or v-CRK. We show that individual domains of CAS associate with specific subsets of proteins in vitro, and that many of these interactions are dependent on the state of tyrosine-phosphorylation of CAS. Sequences necessary for interacting with the focal adhesion kinase pp125FAK (FAK), v-SRC and v-CRK have been mapped to distinct regions of CAS. In addition, the identification of a number of putative CAS-binding partners that are present in crk-transformed cell extracts but undetectable in normal and src-transformed cell extracts supports a model in which unique protein complexes are formed in response to different signals.

Calmodulin regulates the disassembly of cortical F-actin in mast cells but is not required for secretion.

Secretion is dependent on a rise in cytosolic Ca(2+)concentration and is associated with dramatic changes in actin organization. The actin cortex may act as a barrier between secretory vesicles and plasma membrane. Thus, disassembly of this cortex should precede late steps of exocytosis. Here we investigate regulation of both the actin cytoskeleton and secretion by calmodulin. Ca(2+), together with ATP, induces cortical F-actin disassembly in permeabilized rat peritoneal mast cells. This effect is strongly inhibited by removing endogenous calmodulin (using calmodulin inhibitory peptides), and increased by exogenous calmodulin. Neither treatment, however, affects secretion. Low concentrations ( approximately 1 microM) of a specific inhibitor of myosin light chain kinase, ML-7, prevent F-actin disassembly, but not secretion. In contrast, a myosin inhibitor affecting both conventional and unconventional myosins, BDM, decreases cortical disassembly as well as secretion. Observations of fluorescein-calmodulin, introduced into permeabilized cells, confirmed a strong (Ca(2+)-independent) association of calmodulin with the actin cortex. In addition, fluorescein-calmodulin enters the nuclei in a Ca(2+)-dependent manner. In conclusion, calmodulin promotes myosin II-based contraction of the membrane cytoskeleton, which is a prerequisite for its disassembly. The late steps of exocytosis, however, require neither calmodulin nor cortical F-actin disassembly, but may be modulated by unconventional myosin(s).

Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo.

Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.

Oxidative stress is involved in seizure-induced neurodegeneration in the kindling model of epilepsy.

Reactive oxygen species have been implicated in the development of seizures under pathological conditions and linked to seizure-induced neurodegeneration. There has been little direct evidence, however, of free radical production resulting from seizures. Using amygdala-kindled rats, we have examined the generation of reactive oxygen species following seizures, and their possible contribution to seizure development and seizure-induced neuronal loss. The concentrations of two products of free radical-induced lipid peroxidation, malonaldehyde and 4-hydroxy-2(E)-nonenal, were measured using colorimetric assays. Lipid peroxidation was increased in both hemispheres of kindled rats as compared to sham-operated controls. Cell death was also significantly increased in all hippocampal areas. Antioxidants (vitamin E and glutathione) prevented the rise in lipid peroxides and hippocampal neuronal death during kindling, but did not arrest the development of seizures.Thus, epileptiform activity can result in free radical production which may be one of the factors leading to cell death.

Suppression of K(+)-induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium-derived hyperpolarizing factor.

In intact mesenteric arteries, increasing [K(+)]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 microM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K(+)]o. In endothelium-denuded vessels, increasing [K(+)]o by 5 mM hyperpolarized the smooth muscle but K(+) had no effect after depolarization by 10 microM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K(+) was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K(+)-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K(+) via smooth muscle K(+) channels produces a local increase in [K(+)]o which impairs repolarization to added K(+). Thus, studies involving vessels precontracted with agonists which increase [K(+)]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K(+).

K+-induced hyperpolarization in rat mesenteric artery: identification, localization and role of Na+/K+-ATPases.

1. Mechanisms underlying K(+)-induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium-denuded rat mesenteric arteries (for all mean values, n=4). 2. Myocyte resting membrane potential (m.p.) was -58.8+/-0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6+/-0.7 mV) or presence (15.8+/-1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 microM barium, hyperpolarization to 5 mM KCl was essentially abolished. 3. In the presence of 10 microM phenylephrine (m.p. -33.7+/-3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4-aminopyridine but was restored (-26.9+/-1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+-induced repolarization was insensitive to barium (30 microM) but abolished by 500 nM ouabain alone. 4. In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K(+) was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 microM glibenclamide. 5. RT-PCR, Western blotting and immunohistochemical techniques collectively showed the presence of alpha(1)-, alpha(2)- and alpha(3)-subunits of Na(+)/K(+)-ATPase in the myocytes. 6. In K(+)-free solution, re-introduction of K(+) (to 4.6 mM) hyperpolarized myocytes by 20.9+/-0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 microM ouabain. 7. We conclude that under basal conditions, Na(+)/K(+)-ATPases containing alpha(2)- and/or alpha(3)-subunits are partially responsible for the observed K(+)-induced effects. The opening of myocyte K(+) channels (by levcromakalim or phenylephrine) creates a 'K(+) cloud' around the cells which fully activates Na(+)/K(+)-ATPase and thereby abolishes further responses to [K(+)](o) elevation.

Functional evidence of a role for two-pore domain potassium channels in rat mesenteric and pulmonary arteries.

1. Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pH(o)) change membrane potential (E(M)) in rat mesenteric and pulmonary arteries. 2. Changing pH(o) from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 microm) or bupivacaine (100 microm) reversed the hyperpolarisation associated with alkaline pH(o), shifting the E(M) of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 microm) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. 3. K(+) channel blockade by 4-aminopyridine (4-AP) (5 mm), tetraethylammonium (TEA) (10 mm), Ba(2+) (30 microm) and glibenclamide (10 microm) depolarised the pulmonary artery. However, shifts in E(M) with changes in pH(o) remained and were sensitive to anandamide (10 microm), bupivacaine (100 microm) or Zn(2+) (200 microm). 4. Anandamide (0.3-60 microm) or bupivacaine (0.3-300 microm) caused a concentration-dependent increase in basal tone in pulmonary arteries. 5. RT-PCR demonstrated the expression of TASK-1, TASK-2, THIK-1, TRAAK, TREK-1, TWIK-1 and TWIK-2 in mesenteric arteries and TASK-1, TASK-2, THIK-1, TREK-2 and TWIK-2 in pulmonary arteries. TASK-1, TASK-2, TREK-1 and TWIK-2 protein was demonstrated in both arteries by immunostaining. 6. These experiments provide evidence for the presence of two-pore domain K(+) channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK-1 channels is most likely to have mediated the pH-induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.

Characterization of an apamin-sensitive small-conductance Ca(2+)-activated K(+) channel in porcine coronary artery endothelium: relevance to EDHF.

1. The apamin-sensitive small-conductance Ca(2+)-activated K(+) channel (SK(Ca)) was characterized in porcine coronary arteries. 2. In intact arteries, 100 nM substance P and 600 microM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8 +/- 0.8 mV and 24.1 +/- 1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8 +/- 0.3 mV). 3. In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K(+) conductance of 6.8 +/- 0.04 pS. The open-probability was increased by Ca(2+) and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7 +/- 0.03 pS inhibited by d-tubocurarine was observed infrequently. 4. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SK(Ca) was detected by RT - PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6. In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SK(Ca) containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.