PubMed Health⌕ Search

Biomedical subjects

S P Hardy

Publications and source records attributed to S P Hardy.

At least 19 recordsLinked to original sources
PubMed2000

Membrane impermeant antioestrogens discriminate between ligand- and voltage-gated cation channels in NG108-15 cells.

Native 5-HT(3) and AChR ligand-gated cation channels can be inhibited (blocked) by the non-steroidal antioestrogen tamoxifen. However, the exact site and mechanism of inhibition by tamoxifen on these channels remain unclear. We have investigated the action of the membrane impermeant quaternary derivative, ethylbromide tamoxifen (EBT), on native ligand-gated 5-HT(3) receptor channels and voltage-gated K(+) channels in NG108-15 cells using whole cell patch clamp. Extracellular EBT inhibited whole cell cationic currents of 5-HT(3) receptors with IC(50) of 0.22+/-0.4 microM (n(H)=1.05+/-0.2). The channel block was characterised by voltage independent and use independent behaviour (similar to that of tamoxifen). EBT was unable to inhibit voltage-gated K(+) currents in NG108-15 cells. This was in contrast to the inhibition by tamoxifen which, at similar concentrations, accelerated the apparent inactivation of these outward K(+) currents. The inhibition of 5-HT(3) receptors by a membrane impermeant derivative of tamoxifen supports the view that the binding site for antioestrogens is extracellular and the inhibition is not mediated through genomic/transcriptional activity.

Animals↗
PubMed2000

Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines.

The dominant route for Cl(-) secretion in mouse tracheal epithelium is via Cl(-) channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10(-4) M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I(sc)) and resistance of both cell lines, with larger increases in I(sc) being elicited by ionomycin (10(-6) M). Both cell lines expressed P(2)Y(2) receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10(-4) M) with an increase in I(sc). This response could be inhibited by DIDS and was abolished in the presence of Cl(-)-free Ringer solution. Reducing the mucosal Cl(-) concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I(sc) and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl(-) currents and may prove valuable tools in studying the properties of this pathway.

Amiloride↗
PubMed1999

Cationic currents induced by Clostridium perfringens type A enterotoxin in human intestinal CaCO-2 cells.

Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals. The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane. The effect of purified C. perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp. Mucosal application of C. perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa. These changes were independent of extracellular calcium. Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin. Currents were blocked by subsequent exposure to mucosal barium and zinc. Zinc also prevented the development of the current increases in apical membranes. Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings. These data indicate that C. perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions.

Caco-2 Cells↗
PubMed1999

Advantages of percutaneous hollow needle technique for iliac bone harvest in alveolar cleft grafting.

OBJECTIVE: To evaluate the percutaneous hollow needle technique for bone harvest to determine if morbidity from the bone donor site can be reduced significantly. METHODS: A retrospective chart review was performed evaluating all patients undergoing alveolar bone grafting at our institution from January 1992 through December 1996. Patients who underwent additional major procedures were excluded. Group I consisted of 12 patients in whom the percutaneous technique was utilized. The patients had an average age of 11.1 years (range: 8 to 15 years). Six were male and six were female. One had a bilateral cleft. Group II consisted of 15 patients in whom the conventional open technique for iliac crest bone harvest was used. They had an average age of 13.1 years (range: 7 to 31 years). Six were male and nine were female. Two had bilateral clefts. Minimum follow-up was 6 months. We evaluated intraoperative blood loss, total postoperative analgesia requirement, and length of hospital stay based on a retrospective hospital chart review. RESULTS: A significant difference was found between the two groups regarding intraoperative blood loss (group I: 83.3 cm3, group II: 208 cm3; p = .0015), postoperative total analgesia requirement (group I: 0.04 mg/kg, range: 0 to 0.17 mg/kg; group II: 0.34 mg/kg, range: 0.03 to 0.74 mg/kg; p = .0002), and length of hospital stay (group I: 1.0 days, group II: 2.13 days; p = .0001). There was no significant change in these results when bilateral clefts were excluded. CONCLUSION: Iliac bone graft harvest using the percutaneous hollow needle technique results in less blood loss, decreased postoperative pain, and shorter hospital stays compared with the open technique.

Adolescent↗
PubMed1998

Inhibition of voltage-gated cationic channels in rat embryonic hypothalamic neurones and C1300 neuroblastoma cells by triphenylethylene antioestrogens.

The effect of the non-steroidal antioestrogens tamoxifen and toremifene on voltage-gated cationic currents was examined in primary cultures of rat hypothalamic neurones and the C1300 mouse neuroblastoma cell line using the whole-cell patch clamp technique. When applied to the external bathing solution both tamoxifen and toremifene were able to inhibit TTX-sensitive sodium currents with IC50 values of 1-2 microM and delayed rectifier type potassium currents (IC50, 2-3 microM). However, only toremifene showed a significant inhibition of the I(A) current (IC50 3 microM). Inhibition of voltage-gated cationic currents was significantly impaired when tamoxifen was applied in a serum-containing solution. The steroidal antioestrogen ICI 182,780 did not inhibit any of the currents at 10 microM.

Animals↗
PubMed1998

Inhibition of ligand-gated cation-selective channels by tamoxifen.

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.

Animals↗
PubMed1997

Cycling Werner's syndrome fibroblasts display calcium-dependent potassium currents.

Werner's Syndrome (WS) fibroblasts undergo premature senescence. Two hypotheses have been proposed to explain this phenomenon: (i) the phenotype is due to the overexpression of senescence-specific proteins in every cell in the population. Such proteins are known to suppress calcium-dependent potassium currents. (ii) The WS mutation greatly increases the proportion of cells that stop cycling at each generation and become senescent. If hypothesis (i) is correct, such currents should be suppressed in all WS fibroblasts; whereas hypothesis (ii) predicts that they will be retained in the cycling fraction of the population. To distinguish between these hypotheses whole-cell patch-clamp currents were recorded from cycling cells. Slowly activating outward calcium-dependent potassium currents were detected in both cycling WS and control fibroblasts. These findings support hypothesis (ii): the premature senescence of WS fibroblasts is due to an increased rate of transition from cycling to senescence in the total cell population.

Calcium↗
PubMed1997

Ocular staphyloma associated with facial clefting.

A staphyloma is an uncommon ocular lesion consisting of an attenuation in the sclera, which, along with the underlying uveal tissue, bulges to form a raised pigmented area on the eye. The scleral defect predisposes the globe to rupture under conditions of increased intraocular pressure, which might occur while retracting the eye during cranio-orbital surgery. We report a case of a staphyloma in a child with bilateral facial clefts. Before hypertelorism correction, she underwent scleral repair with a cadaveric graft. Her orbital repositioning was performed without incident 10 months later. The significance of a possible association between facial clefting and staphyloma is discussed.

Cadaver↗
PubMed1996

The multidrug resistance P-glycoprotein modulates cell regulatory volume decrease.

Cell volume is frequently down-regulated by the activation of anion channels. The role of cell swelling-activated chloride channels in cell volume regulation has been studied using the patch-clamp technique and a non-invasive microspectrofluorimetric assay for changes in cell volume. The rate of activation of these chloride channels was shown to limit the rate of regulatory volume decrease (RVD) in response to hyposmotic solutions. Expression of the human MDR1 or mouse mdr1a genes, but not the mouse mdr1b gene, encoding the multidrug resistance P-glycoprotein (P-gp), increased the rate of channel activation and the rate of RVD. In addition, P-gp decreased the magnitude of hyposmotic shock required to activate the channels and to elicit RVD. Tamoxifen selectively inhibited both chloride channel activity and RVD. No effect on potassium channel activity was elicited by expression of P-gp. The data show that, in these cell types, swelling-activated chloride channels have a central role in RVD. Moreover, they clarify the role of P-gp in channel activation and provide direct evidence that P-gp, through its effect on chloride channel activation, enhances the ability of cells to down-regulate their volume.

3T3 Cells↗
PubMed1995

Protein kinase C-mediated phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels.

The multidrug resistance P-glycoprotein (P-gp), which transports hydrophobic drugs out of cells, is also associated with volume-activated chloride currents. It is not yet clear whether P-gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P-gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume-activated chloride currents indistinguishable from those obtained in P-gp-expressing cells except that they were insensitive to PKC. HeLa cells did not express detectable P-gp but, following transient transfection with cDNA encoding P-gp, the volume-activated channels acquired PKC regulation. PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P-gp were replaced with alanine. Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation. These data demonstrate that PKC-mediated phosphorylation of P-gp regulates the activity of an endogenous chloride channel and thus indicate that P-gp is a channel regulator.

3T3 Cells↗
PubMed1995

Chloride channels: a state of flux.

Chloride channels play important functions in different aspects of cell physiology including volume regulation, transepithelial ion transport and stabilization of membrane potential. In recent years the molecular identity of the chloride channels defective in cystic fibrosis and myotonia congenita has been elucidated, highlighting the importance of anion-selective channels in cell and tissue function. Concurrently, several proteins have been identified as chloride channels along with proteins that possess channel regulatory behavior. Novel interactions with more potent pharmacological compounds have been reported with different chloride channels. This burgeoning field of interest is reviewed.

Animals↗
PubMed1995

Lens opacification by antioestrogens: tamoxifen vs ICI 182,780.

The antioestrogen, tamoxifen, blocks volume-regulated chloride channels and reduces transparency in bovine lenses maintained in vitro. In contrast to tamoxifen, the steroidal antioestrogen, ICI 182780, did not block volume-regulated chloride currents in three cultured cell lines and required 10 fold higher concentration to induce significant opacification of bovine lenses maintained in vitro. These data suggest that ocular toxic side effects will be minimized by use of the steroidal (ICI 182780) rather than nonsteroidal antioestrogens (tamoxifen).

Animals↗
PubMed1994

Novel plasma membrane action of estrogen and antiestrogens revealed by their regulation of a large conductance chloride channel.

Antiestrogens antagonize many genomic effects of estrogen through binding to the nuclear estrogen receptor. We report here that NIH3T3 fibroblasts grown in the presence of colchicine acquire the activation of a large conductance chloride channel upon exposure to extracellular but not intracellular antiestrogens. This effect can be prevented by extracellular 17 beta-estradiol, but not intracellular 17 beta-estradiol or extracellular 17 alpha-estradiol. This is the first demonstration of a regulatory role for antiestrogens and estrogens in the regulation of ionic channels occurring through an interaction of these compounds with a plasma membrane binding site distinct from the classical estrogen receptor and subsequent activation of intracellular second messenger pathway (or pathways).

3T3 Cells↗
PubMed1994

Tamoxifen blocks chloride channels. A possible mechanism for cataract formation.

Tamoxifen is an antiestrogen frequently used in the treatment of breast cancer and is currently being assessed as a prophylactic for those at high risk of developing tumors. We have found that tamoxifen and its derivatives are high-affinity blockers of specific chloride channels. This blockade appears to be independent of the interaction of tamoxifen with the estrogen receptor and therefore reflects an alternative cellular target. One of the clinical side effects of tamoxifen is impaired vision and cataract. Chloride channels in the lens of the eye were shown to be essential for maintaining normal lens hydration and transmittance. These channels were blocked by tamoxifen and, in organ culture, tamoxifen led to lens opacity associated with cataracts at clinically relevant concentrations. These data suggest a molecular mechanism by which tamoxifen can cause cataract formation and have implications for the clinical use of tamoxifen and related antiestrogens.

3T3 Cells↗
PubMed1994

Regulation of volume activated chloride channels by protein kinase C-mediated phosphorylation of P-glycoprotein.

The multidrug resistance P-glycoprotein (Pgp) transports hydrophobic drugs out of cells and has been recently associated with volume-activated chloride channels. Activation of these channels by hypotonic swelling was seen to be prevented by protein kinase C (PKC) in cells expressing high levels of Pgp by transfection. HeLa cells possess equivalent chloride currents yet they are not regulated by PKC. HeLa cells do not express Pgp as assessed by Western blotting. Following transfection of HeLa cells with cDNA encoding for Pgp, PKC-dependent suppression of volume activated chloride currents was observed. PKC regulation in transiently transfected HeLa cells was abolished by alanine replacement of the serine/threonine residues in the consensus phosphorylation sites of the linker region of Pgp. Replacement of these residues with glutamate, to mimic the effect of phosphorylation, mimicked the effects of PKC on channel activation. These results indicate that overexpression of Pgp confers PKC-regulation of endogenous volume-activated chloride channels. More generally they favour a model in which Pgp acts as a regulator of volume-activated chloride channels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Pain after subcutaneous injection of recombinant human erythropoietin: does Emla cream help?

Recombinant human erythropoietin (rHuEpo) is used to correct anaemia in dialysis patients. Subcutaneous administration of rHuEpo may be associated with pain at the injection site. This study assessed the pain of subcutaneous infiltration of two different preparations of rHuEpo, alpha and beta, and the value of a local anaesthetic (Emla) cream, in reducing the pain of infiltration. Forty-eight haemodialysis patients were enrolled into a double-blind, placebo-controlled, paired-comparison study. Pain was assessed using a visual analogue scale, a verbal descriptive scale and a direct comparison between paired treatments. Subcutaneous injection of rHuEpo alpha was more painful than rHuEpo beta (P < 0.001); using placebo cream 42% of patients described the pain of rHuEpo alpha as severe or very severe, whereas none of the patients found rHuEpo beta so painful. Application of Emla for at least 2 h prior to injection resulted in a significant reduction in the pain of both preparations, but was unable to reduce the pain of rHuEpo alpha to that of rHuEpo beta. Subcutaneous injection of rHuEpo alpha is more painful than rHuEpo beta, even after application of Emla. Although the discomfort of rHuEpo beta is graded as very mild by most adult patients the use of Emla is associated with a significant reduction in discomfort, which may be of benefit to paediatric patients.

Adult↗
PubMed1993

Nipple-areola reconstruction with intradermal tattoo and double-opposing pennant flaps.

Reconstruction of the nipple-areolar complex is an important part of postmastectomy breast reconstruction. Intradermal tattooing is effective for creating areolar pigmentation and is simpler than skin grafting. However, a simple method for the construction of a normal nipple using local tissues remains a challenge. Several techniques achieve long-term nipple projection but at the expense of complicated flap design, multidirectional scars, and often a need for skin grafting to obtain a uniform-appearing areola. Our one-stage method of nipple-areola reconstruction uses intradermal tattooing for pigmentation and double-opposing pennant flaps for nipple reconstruction. The entire procedure is simple and fast, routinely performed in < 30 minutes under local anaesthesia in the office. From September 1989 to March 1992 we performed 102 reconstructions. The method produces a realistic appearing nipple. We have had no flap necrosis.

Female↗