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Sodium influx inhibitors are vasodilator and antihypertensive agents
J Mel
Cell
Cardiol
22 (Supplement
I) (1990)
P61
EDRF SYNTHETASE IN BOVINE AORTA ENDOTHELIAL CELLS AND NlE-115 NEUROBLASTOMA CELLS: SUBCELLULAR LOCALIZATION AND REGULATION BY CALCIUY AND CALYODULIN Midlad Hdler, Ukkh F6ratarmpnn. Las D.GoMy. Kunb bhii. Jan&r S.Po&ck, Harald H.H.W.S&mld and Farid Murad. Abbott Labs, Ab4ctl Park, II. 50054 snd Nwthwaatam univ. Cwcaw. II. 50511 USA. Bovlno amlc ecddhdlal calls (BAE) uld m.sfne ne~mblaatcma cslla (Nl E-l 15) produce subatanca(a) wkh pharmacological properUrn of andclhallumdwlvad relaxing famcr (EDRF). A bbawy which maaauaa allmulti d Alma mylala cydasa (sGC) in fetal rat kmg flbmblasta (RFL-6) WPI davabpad to monkor ha EDRF &vily. BAE ah ralaaaa EDRF ho dw me&m uhkh increased cGMP levels in the RFL-5 calls. Bradyidnin. thromth or ADP and nwmtansin anhanmd hwmalbn ol EDRF by BAE and NlE-115 cells. raapeaivaly. In Ca2+ - free madum lha hormonal slnwlatkm dlwppaars. ln Nl E-l 15 odla, EDRF la pm&cad axdusively In the cylosd whereas In BAE calls some cl the aclMly was found also h the panlarlata fraclkm. Suparcxidn dsmulaaa (SOD) Is needed for assessing of EDRF prcductlon. Only the cytoaclic enzyme(s) are aandtlva lo Ca2+ al 0.1 QSmM. NADPH and L-arginine supported EDRF iormailon. NG&ro L-arglnine or NG-mcncmalhyl L-arglnina ccmpalltlvely inhibited EDRF synlhesir froml-arginine. Hemoglobin or methylene blue blcckad the affacts of EDRF. Cydo-oxyganaaa nd lfpoxyganaaa Inhibitor8 (indcmachadn and gossypol, respedivdy) had only liltle affect The aGC _ aUnwlaUng acMfy of tie muyma pfqrdkm wan hhlbHad by eradddcnk add excluding a lipid paroxidaflon pro&cl to account fa sGC sllmuladon. Synllwalr of EDRF vu Inhibited by cafmodulin inhibitors, e.g.. calmfdazolium (IC50=10pM). ldflwparazine (1OpM). fandilln (8OpM), W-7 (12OpM) and ccn’qfnmd 4VSO (3pgM). These inhibitors had no effacf (except fandllin al higher cone) cm acdium nitroprursida (SNP) stknul~ In RFL-6 datactw calls. Frtination of the Nl E-l 15 cyicscl on DE-52 and eltion wilh KCI yielded two protein fraolbna aluting al 0.1 M and 0.3M. Each fraction alone did no1 suppon EDRF synthesir from L-arginlne + NADPH. CornMIng bolh fradkmr raamrad EDRF synthaals. Tha aacwd fractkn could be replaced by calmo&lln. In lhesa experimen(s caltidazdium and conpound 43450 inhibited the ma&on. ll may be concluded that BAE and NlE-115 calls synthedze EDRF from L-arginine or a r&tad conpound which saams 10 be NO ratha than a product of lipid peroxidalion. Tha piocasa require a rnftux of and activatfon by Ca2+. Tha cylosdlc enzyme ia regulated by caJrr!c&lin and Ca2+. The parBculata BAE enzyme may -nl for basal prrx&Licn of EDRF in endolhdal calls. SupporIad in parl by Research Grants AM 30757. HL 28474and NRSA grant AR 08080 from he Nationa! Institutes of Health.
p62
SODIUM Francis
INFLUX INHIBITORS ABE VASODILATOR AND ANTIHYPERTENSIVE AGENTS. J. Haddy, andMotila1 B. Pamnani. Department of Physiology, Uniformed Services University, Bethesda, MD 20814-4799. In theory, Na+ influx inhibitors should be vasodilators. Furthermore, they should be most effective in those models of hypertension characterized by increased membrane permeability to sodium. We have tested these hypotheses by injecting amiloride and 14 of its analogues into the dog forelimb and administering one of the analogues to four different models of hypertension. In the anesthetized dog, intrabrachial injection of amiloride and 7 of its analogues produced vasodilatation. The only analogue with greater vasodilator activity than amiloride was 6-iodo-amilordie. Analogues specific for Na’-H’ exchange blockade were inactive. Intravenous administration of 6-iodoamiforide produced a prompt large sustained decrease in blood pressure in anesthetized rats with genetic hypertension characterized by increased VSHC sodium permeability (SHR: Dahl S) but only a transient decrease inblood pressure in rats with investigator induced hypertension without increased VSMC sodium permeability (one-kidney, one clip; reducedrenalmass-saline). Oraladministrationalso loweredbloodpressure chronically in the genetic models. The antihypertensive effect was not the result of natriuresis and diuresis. We presume that it results fromvasodilatation since the agenthyperpolarizes VSMC's in isolated arteries andhas no effect on isolatedhearts. Presumablyhyperpolarization reduced calcium permeability and hence calcium influx. The data presented support the two hypotheses.
P63
QUANTITATI~N 0~ CARDIAC ~~CYTE DIMENSIONS AND NUMBER IN SPONTANE~IJSLY HYPERTENSIVE RATS TREATED WITH CALCIUM ANTAGONISTS. Scott E. Campbell, Karel Rakusan, Zdenek Turek, Stanislav Kazda. Departments of Physiology, University of Ottawa, Ottawa, Canada and Catholic University, Nijmegpn, Netherlands and Bayer AG, Wuppertal, FRG. J'revious histological analysis of cardiac tissue from nisoldipine-treated SHR indicated that myocyte hyperplasia may accompany capillary proliferation. To determine whether cardiac myocyte hyperplasia was associated with calcium antagonist treatment for hypertension, SHR and WKY rats were fed rat chow that was normal, or containing nifedipine (NIF; 1000 ppm) or nisoldipine (NIS; 1000 ppm). Isolated myocytes were prepared by retrograde coronary perfusion with collagenase after 22 weeks of trratmerit . Blood pressure (BP) was determined from carotid cannulation. Cell volume (CV) cell length (CL) was direcr.ly measured and was measured with a Coulter Channelyzer, cross-sectional area was estimated from CV/CL. Cell number was calculated for both ventricles. Heart weight to body weight ratios (RW/BW) were greater (~'0.01) in SHR groups compared to WKY controls. HW/BW was decreased in both NIF (p
Cell
Cardiol
22 (Supplement
I) (1990)
P61
EDRF SYNTHETASE IN BOVINE AORTA ENDOTHELIAL CELLS AND NlE-115 NEUROBLASTOMA CELLS: SUBCELLULAR LOCALIZATION AND REGULATION BY CALCIUY AND CALYODULIN Midlad Hdler, Ukkh F6ratarmpnn. Las D.GoMy. Kunb bhii. Jan&r S.Po&ck, Harald H.H.W.S&mld and Farid Murad. Abbott Labs, Ab4ctl Park, II. 50054 snd Nwthwaatam univ. Cwcaw. II. 50511 USA. Bovlno amlc ecddhdlal calls (BAE) uld m.sfne ne~mblaatcma cslla (Nl E-l 15) produce subatanca(a) wkh pharmacological properUrn of andclhallumdwlvad relaxing famcr (EDRF). A bbawy which maaauaa allmulti d Alma mylala cydasa (sGC) in fetal rat kmg flbmblasta (RFL-6) WPI davabpad to monkor ha EDRF &vily. BAE ah ralaaaa EDRF ho dw me&m uhkh increased cGMP levels in the RFL-5 calls. Bradyidnin. thromth or ADP and nwmtansin anhanmd hwmalbn ol EDRF by BAE and NlE-115 cells. raapeaivaly. In Ca2+ - free madum lha hormonal slnwlatkm dlwppaars. ln Nl E-l 15 odla, EDRF la pm&cad axdusively In the cylosd whereas In BAE calls some cl the aclMly was found also h the panlarlata fraclkm. Suparcxidn dsmulaaa (SOD) Is needed for assessing of EDRF prcductlon. Only the cytoaclic enzyme(s) are aandtlva lo Ca2+ al 0.1 QSmM. NADPH and L-arginine supported EDRF iormailon. NG&ro L-arglnine or NG-mcncmalhyl L-arglnina ccmpalltlvely inhibited EDRF synlhesir froml-arginine. Hemoglobin or methylene blue blcckad the affacts of EDRF. Cydo-oxyganaaa nd lfpoxyganaaa Inhibitor8 (indcmachadn and gossypol, respedivdy) had only liltle affect The aGC _ aUnwlaUng acMfy of tie muyma pfqrdkm wan hhlbHad by eradddcnk add excluding a lipid paroxidaflon pro&cl to account fa sGC sllmuladon. Synllwalr of EDRF vu Inhibited by cafmodulin inhibitors, e.g.. calmfdazolium (IC50=10pM). ldflwparazine (1OpM). fandilln (8OpM), W-7 (12OpM) and ccn’qfnmd 4VSO (3pgM). These inhibitors had no effacf (except fandllin al higher cone) cm acdium nitroprursida (SNP) stknul~ In RFL-6 datactw calls. Frtination of the Nl E-l 15 cyicscl on DE-52 and eltion wilh KCI yielded two protein fraolbna aluting al 0.1 M and 0.3M. Each fraction alone did no1 suppon EDRF synthesir from L-arginlne + NADPH. CornMIng bolh fradkmr raamrad EDRF synthaals. Tha aacwd fractkn could be replaced by calmo&lln. In lhesa experimen(s caltidazdium and conpound 43450 inhibited the ma&on. ll may be concluded that BAE and NlE-115 calls synthedze EDRF from L-arginine or a r&tad conpound which saams 10 be NO ratha than a product of lipid peroxidalion. Tha piocasa require a rnftux of and activatfon by Ca2+. Tha cylosdlc enzyme ia regulated by caJrr!c&lin and Ca2+. The parBculata BAE enzyme may -nl for basal prrx&Licn of EDRF in endolhdal calls. SupporIad in parl by Research Grants AM 30757. HL 28474and NRSA grant AR 08080 from he Nationa! Institutes of Health.
p62
SODIUM Francis
INFLUX INHIBITORS ABE VASODILATOR AND ANTIHYPERTENSIVE AGENTS. J. Haddy, andMotila1 B. Pamnani. Department of Physiology, Uniformed Services University, Bethesda, MD 20814-4799. In theory, Na+ influx inhibitors should be vasodilators. Furthermore, they should be most effective in those models of hypertension characterized by increased membrane permeability to sodium. We have tested these hypotheses by injecting amiloride and 14 of its analogues into the dog forelimb and administering one of the analogues to four different models of hypertension. In the anesthetized dog, intrabrachial injection of amiloride and 7 of its analogues produced vasodilatation. The only analogue with greater vasodilator activity than amiloride was 6-iodo-amilordie. Analogues specific for Na’-H’ exchange blockade were inactive. Intravenous administration of 6-iodoamiforide produced a prompt large sustained decrease in blood pressure in anesthetized rats with genetic hypertension characterized by increased VSHC sodium permeability (SHR: Dahl S) but only a transient decrease inblood pressure in rats with investigator induced hypertension without increased VSMC sodium permeability (one-kidney, one clip; reducedrenalmass-saline). Oraladministrationalso loweredbloodpressure chronically in the genetic models. The antihypertensive effect was not the result of natriuresis and diuresis. We presume that it results fromvasodilatation since the agenthyperpolarizes VSMC's in isolated arteries andhas no effect on isolatedhearts. Presumablyhyperpolarization reduced calcium permeability and hence calcium influx. The data presented support the two hypotheses.
P63
QUANTITATI~N 0~ CARDIAC ~~CYTE DIMENSIONS AND NUMBER IN SPONTANE~IJSLY HYPERTENSIVE RATS TREATED WITH CALCIUM ANTAGONISTS. Scott E. Campbell, Karel Rakusan, Zdenek Turek, Stanislav Kazda. Departments of Physiology, University of Ottawa, Ottawa, Canada and Catholic University, Nijmegpn, Netherlands and Bayer AG, Wuppertal, FRG. J'revious histological analysis of cardiac tissue from nisoldipine-treated SHR indicated that myocyte hyperplasia may accompany capillary proliferation. To determine whether cardiac myocyte hyperplasia was associated with calcium antagonist treatment for hypertension, SHR and WKY rats were fed rat chow that was normal, or containing nifedipine (NIF; 1000 ppm) or nisoldipine (NIS; 1000 ppm). Isolated myocytes were prepared by retrograde coronary perfusion with collagenase after 22 weeks of trratmerit . Blood pressure (BP) was determined from carotid cannulation. Cell volume (CV) cell length (CL) was direcr.ly measured and was measured with a Coulter Channelyzer, cross-sectional area was estimated from CV/CL. Cell number was calculated for both ventricles. Heart weight to body weight ratios (RW/BW) were greater (~'0.01) in SHR groups compared to WKY controls. HW/BW was decreased in both NIF (p