Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells

Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calci...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:The Journal of clinical investigation 1992-08, Vol.90 (2), p.429-438
Hauptverfasser: GESEK, F. A, FRIEDMAN, P. A
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
container_end_page 438
container_issue 2
container_start_page 429
container_title The Journal of clinical investigation
container_volume 90
creator GESEK, F. A
FRIEDMAN, P. A
description Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.
doi_str_mv 10.1172/jci115878
format Article
fullrecord <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_443118</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>16393302</sourcerecordid><originalsourceid>FETCH-LOGICAL-c535t-d46f0f791713c44bb9a4396ab68d8ddb7ed00d88469c5e8529ffce88ac9864333</originalsourceid><addsrcrecordid>eNqFkU1P3DAQhq2qCBbaQ39AJR8qJA4BO-Mk9qEHtOJjEYhLe7Yc2-l65cTb2EGCX49XWQE99TSH95nRvHoQ-kbJOaVNebHRjtKKN_wTWtCq4gUvgX9GC0JKWogG-BE6jnFDCGWsYofokEJZChALpB-sXqvBxR6HDmvltZt6nEY1xG0YE47J9ZNXyRrcPmO99mEMae3UizMWuwH3YYoWGxeT8liH4Sn4aQenqZ28xdp6H7-gg075aL_u5wn6fX31a3lb3D_erJaX94WuoEqFYXVHukbQhoJmrG2FYiBq1dbccGPaxhpCDOesFrqyvCpF12nLudKC1wwATtDP-e52antrtB1yDy-3o-vV-CyDcvLfZHBr-Sc8ScaAUp73T_f7Y_g72Zhk7-KugRpsrikboKTmdfNfkNYgAEiZwbMZ1GOIcbTd2zOUyJ05ebdczeYy-_3j9-_krCrnP_a5itlTlxVpF98wBiVALvEKxgqjUg</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>16393302</pqid></control><display><type>article</type><title>Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells</title><source>MEDLINE</source><source>EZB-FREE-00999 freely available EZB journals</source><source>PubMed Central</source><source>Alma/SFX Local Collection</source><creator>GESEK, F. A ; FRIEDMAN, P. A</creator><creatorcontrib>GESEK, F. A ; FRIEDMAN, P. A</creatorcontrib><description>Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.</description><identifier>ISSN: 0021-9738</identifier><identifier>EISSN: 1558-8238</identifier><identifier>DOI: 10.1172/jci115878</identifier><identifier>PMID: 1322939</identifier><identifier>CODEN: JCINAO</identifier><language>eng</language><publisher>Ann Arbor, MI: American Society for Clinical Investigation</publisher><subject>Animals ; Biological and medical sciences ; Biological Transport - drug effects ; Bumetanide - pharmacology ; calcium ; Calcium - metabolism ; cells ; Cells, Cultured ; Chloride Channels ; Chlorides - metabolism ; chlorothiazide ; Chlorothiazide - pharmacology ; distal tubules ; In Vitro Techniques ; kidney ; Kidney Tubules, Distal - drug effects ; Medical sciences ; Membrane Potentials - drug effects ; Membrane Proteins - drug effects ; Mice ; Nifedipine - pharmacology ; Nitrobenzoates - pharmacology ; Pharmacology. Drug treatments ; Sodium - metabolism ; stimulation ; transport ; Urinary system</subject><ispartof>The Journal of clinical investigation, 1992-08, Vol.90 (2), p.429-438</ispartof><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c535t-d46f0f791713c44bb9a4396ab68d8ddb7ed00d88469c5e8529ffce88ac9864333</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC443118/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC443118/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&amp;idt=4323318$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1322939$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>GESEK, F. A</creatorcontrib><creatorcontrib>FRIEDMAN, P. A</creatorcontrib><title>Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells</title><title>The Journal of clinical investigation</title><addtitle>J Clin Invest</addtitle><description>Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Biological Transport - drug effects</subject><subject>Bumetanide - pharmacology</subject><subject>calcium</subject><subject>Calcium - metabolism</subject><subject>cells</subject><subject>Cells, Cultured</subject><subject>Chloride Channels</subject><subject>Chlorides - metabolism</subject><subject>chlorothiazide</subject><subject>Chlorothiazide - pharmacology</subject><subject>distal tubules</subject><subject>In Vitro Techniques</subject><subject>kidney</subject><subject>Kidney Tubules, Distal - drug effects</subject><subject>Medical sciences</subject><subject>Membrane Potentials - drug effects</subject><subject>Membrane Proteins - drug effects</subject><subject>Mice</subject><subject>Nifedipine - pharmacology</subject><subject>Nitrobenzoates - pharmacology</subject><subject>Pharmacology. Drug treatments</subject><subject>Sodium - metabolism</subject><subject>stimulation</subject><subject>transport</subject><subject>Urinary system</subject><issn>0021-9738</issn><issn>1558-8238</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1992</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1P3DAQhq2qCBbaQ39AJR8qJA4BO-Mk9qEHtOJjEYhLe7Yc2-l65cTb2EGCX49XWQE99TSH95nRvHoQ-kbJOaVNebHRjtKKN_wTWtCq4gUvgX9GC0JKWogG-BE6jnFDCGWsYofokEJZChALpB-sXqvBxR6HDmvltZt6nEY1xG0YE47J9ZNXyRrcPmO99mEMae3UizMWuwH3YYoWGxeT8liH4Sn4aQenqZ28xdp6H7-gg075aL_u5wn6fX31a3lb3D_erJaX94WuoEqFYXVHukbQhoJmrG2FYiBq1dbccGPaxhpCDOesFrqyvCpF12nLudKC1wwATtDP-e52antrtB1yDy-3o-vV-CyDcvLfZHBr-Sc8ScaAUp73T_f7Y_g72Zhk7-KugRpsrikboKTmdfNfkNYgAEiZwbMZ1GOIcbTd2zOUyJ05ebdczeYy-_3j9-_krCrnP_a5itlTlxVpF98wBiVALvEKxgqjUg</recordid><startdate>19920801</startdate><enddate>19920801</enddate><creator>GESEK, F. A</creator><creator>FRIEDMAN, P. A</creator><general>American Society for Clinical Investigation</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>8FD</scope><scope>FR3</scope><scope>M7Z</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19920801</creationdate><title>Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells</title><author>GESEK, F. A ; FRIEDMAN, P. A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c535t-d46f0f791713c44bb9a4396ab68d8ddb7ed00d88469c5e8529ffce88ac9864333</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1992</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Biological Transport - drug effects</topic><topic>Bumetanide - pharmacology</topic><topic>calcium</topic><topic>Calcium - metabolism</topic><topic>cells</topic><topic>Cells, Cultured</topic><topic>Chloride Channels</topic><topic>Chlorides - metabolism</topic><topic>chlorothiazide</topic><topic>Chlorothiazide - pharmacology</topic><topic>distal tubules</topic><topic>In Vitro Techniques</topic><topic>kidney</topic><topic>Kidney Tubules, Distal - drug effects</topic><topic>Medical sciences</topic><topic>Membrane Potentials - drug effects</topic><topic>Membrane Proteins - drug effects</topic><topic>Mice</topic><topic>Nifedipine - pharmacology</topic><topic>Nitrobenzoates - pharmacology</topic><topic>Pharmacology. Drug treatments</topic><topic>Sodium - metabolism</topic><topic>stimulation</topic><topic>transport</topic><topic>Urinary system</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>GESEK, F. A</creatorcontrib><creatorcontrib>FRIEDMAN, P. A</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium &amp; Calcified Tissue Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of clinical investigation</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>GESEK, F. A</au><au>FRIEDMAN, P. A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells</atitle><jtitle>The Journal of clinical investigation</jtitle><addtitle>J Clin Invest</addtitle><date>1992-08-01</date><risdate>1992</risdate><volume>90</volume><issue>2</issue><spage>429</spage><epage>438</epage><pages>429-438</pages><issn>0021-9738</issn><eissn>1558-8238</eissn><coden>JCINAO</coden><abstract>Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.</abstract><cop>Ann Arbor, MI</cop><pub>American Society for Clinical Investigation</pub><pmid>1322939</pmid><doi>10.1172/jci115878</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record>
fulltext fulltext
identifier ISSN: 0021-9738
ispartof The Journal of clinical investigation, 1992-08, Vol.90 (2), p.429-438
issn 0021-9738
1558-8238
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_443118
source MEDLINE; EZB-FREE-00999 freely available EZB journals; PubMed Central; Alma/SFX Local Collection
subjects Animals
Biological and medical sciences
Biological Transport - drug effects
Bumetanide - pharmacology
calcium
Calcium - metabolism
cells
Cells, Cultured
Chloride Channels
Chlorides - metabolism
chlorothiazide
Chlorothiazide - pharmacology
distal tubules
In Vitro Techniques
kidney
Kidney Tubules, Distal - drug effects
Medical sciences
Membrane Potentials - drug effects
Membrane Proteins - drug effects
Mice
Nifedipine - pharmacology
Nitrobenzoates - pharmacology
Pharmacology. Drug treatments
Sodium - metabolism
stimulation
transport
Urinary system
title Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells
url https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2024-12-24T03%3A57%3A16IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Mechanism%20of%20calcium%20transport%20stimulated%20by%20chlorothiazide%20in%20mouse%20distal%20convoluted%20tubule%20cells&rft.jtitle=The%20Journal%20of%20clinical%20investigation&rft.au=GESEK,%20F.%20A&rft.date=1992-08-01&rft.volume=90&rft.issue=2&rft.spage=429&rft.epage=438&rft.pages=429-438&rft.issn=0021-9738&rft.eissn=1558-8238&rft.coden=JCINAO&rft_id=info:doi/10.1172/jci115878&rft_dat=%3Cproquest_pubme%3E16393302%3C/proquest_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=16393302&rft_id=info:pmid/1322939&rfr_iscdi=true