Biomedical subjects

S P Hardy

Publications and source records attributed to S P Hardy.

12 recordsLinked to original sources

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↗

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↗

PubMed1993

Prolonged carriage of Clostridium difficile in Hirschsprung's disease.

The role of Clostridium difficile in the aetiology of diarrhoea in children with Hirschsprung's disease was investigated in a prospective longitudinal study. In 64 children with Hirschsprung's disease no significant difference was found in the isolation rate of C difficile in patients with diarrhoea (32%) and without diarrhoea (26%). Comparable isolation rates were found in 47 control children with and without diarrhoea (27% and 16% respectively). The number of strains producing toxin B was similar in the four groups of children. In contrast to the disappearance of C difficile by 12 months of age in the control groups of children, C difficile could be repeatedly isolated from a proportion of children with Hirschsprung's disease over 12 months of age. These findings help to reconcile the existing contradictory reports on the incidence of C difficile in Hirschsprung's disease associated enterocolitis.

Adolescent↗

PubMed1993

Electrogenic colonic ion transport in Hirschsprung's disease: reduced secretion to the neural secretagogues acetylcholine and iloprost.

The effects of the abnormal innervation in Hirschsprung's disease on colonic ion transport were examined in vitro using Ussing chambers. The response of the mucosal/submucosal preparations to different secretagogues were investigated in aganglionic and ganglionic rectosigmoid and transverse colon from children with Hirschsprung's disease and compared with normally innervated colon from children with anorectal anomalies. Basal values were similar in aganglionic and ganglionic rectosigmoid colon. Neurally mediated secretion with iloprost (10(-6) M) and acetylcholine (900 and 9 microM) was considerably reduced in aganglionic colon compared with normally innervated ganglionic colon. The ganglionic colon proximal to the aganglionic colon also had a reduced response to acetylcholine despite a normal acetylcholinesterase staining pattern. The responses to Escherichia coli STa enterotoxin (50 MU/ml) and isobutylmethylxanthine (10(-3) M) were similar in ganglionic and aganglionic colon. The response to STa enterotoxin was not changed by the nerve blocking agent tetrodotoxin (10(-6) M). The data show that colonocytes from aganglionic colon are capable of a normal secretory response if stimulated directly by cAMP or cGMP acting secretagogues but secretion in response to neurally mediated secretagogues is impaired. The hypertrophied acetylcholinesterase positive nerve fibres that infiltrate the aganglionic colon are likely to contribute to the reduced secretion to acetylcholine.

1-Methyl-3-isobutylxanthine↗