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Synthetic steriod derivatives as candidates for endogenous digitalis-like factors: Interaction with human placenta digitalis receptors
AJH 1996; 9:69A
POSTERS: Steroids
Kl ALDOSTERONE AND DAY- AND NIGHT TIME AMBULATORY BLOOD PRESSURE PROFILE. H verlooye, D Duprez-, M De Buyzere, and DL Clement-. University Hospital, Gent, Belgium. Both renin and angiotensio II playa key role in blood pressure (BP) regulation during postural changes. As aldosterone (ALD) release is in part determined by other factors, there might be debate whether subsequently ALD levels are related to usual BP. We studied day (09.00 am - 09.00 pm) and night time (09.00 pm - 09.00 am) BP profile in relaton to supine (representative for nIght time) and standing (representative for day time) ALD concentrations in 30 untreated patients (15 men, 15 women, age 47 ± 13 years) with normal or mildly increased ambulatory BP. Ambulatory BP (BP) recordings at 30 min intervals (SpaceLabs 90207) were analysed for day- and night time systolic (SBP), diastolic (OBP) and mean arterial BP (MAP). Table summarizes correlation coefficients (p values) between ABP data and ALD, adjusted for age and BM!. BP ALD (supine) ALD (standing) (mm Hg) (l1.5±7.5 ngldl) (20.9±1l.l ngldl) Day SBP 148±11 0.39 (0.03) 0.50 (0.007) Night SBP 130±14 0.58 (0.001) 0.64 (0.0003) Day MAP 110±9 0.46 (0.01) 0.40 (0.03) Night MAP 96±11 0.60 (0.0009) 0.60 (0.0008) Day OBP 93±9 0.37 (0.05) 0.25 (0.14) Night OBP 78±1l 0.55 (0.003) 0.51 (0.0006) Aldosterone concentrations in supine and standing positions are well correlated with baseline night-time ABP and to a lesser extent with daytime ABP.
Key Words: aldosterone, ambulatory blood pressure, postural changes
K2 SYNTHETIC STEROID DERIVATIVES AS CANDIDATES FOR ENDOGENOUS DIGITALIS·L1KE FACTORS: INTERACTION WITH HUMAN PLACENTA DIGITALIS RECEPTORS. ~ M Sakakibara, P Del Bene, A Uchida. CNR Institute of Clinical Physiology, Plsa, Italy, and Pharm Res Lab Kirln Brewery Co, Gunma, Japan. Allempts to Isolste and chsrscterlze the endogenous digitalis-like factor(s) (EDLF) In mammals have been limited by the small amount of substance typically Isolsted. DigOXin, ouabain, ouabain-like substances, and an ouabain Isomer have been reported as EDLF. On the basis of the molecular weight of Inagaml-Tamura's EDLF and reported EDLF properties, some of us hypothesized, as a possible structure for EDLF, a steroidal skeleton containing a 2,3·catechol structure lacking the lactonlc ring at the 17-posltion of genlns. Several oxygenated estrogen derivatives were hence synthesized and analysed for contractile effect in Isolated rat aorta and guinea pig left atnum, and also many digitoxin dsrlvatives. Aim of this study was to analyse the reactivity of these synthetic compounds to digitalis receptors from human placenta. Using 1251-digoxln as tracer, receptor Interaction was analysed at equilibrium end by a sequential ssturation technique that we found to Incresse the sensitivity of the radioreceptor assay for EDLF detection In plasma. The question whether synthetic compounds Inhibiting digoxin binding acted via the dlgllalls binding site was enalysed by measuring 3H· ouabain binding to placental membranas at various, fixed concenlratlons of the Inhibllor. We found that specific 1251-digoxln binding to placental receptors wss Inhibited by 3 of the 14 compounds tested with ICso ranging between 10 and 10011M. Under the same conditions, ouabain and digoxin were effective at 5.9nM and B.4nM, digitoxigenin and dlgoxlgenln at 30nM, progesterone at 1011M, DHEA·S, cortisol and estradiol st >lOOI1M. Two of 3 synthetic compounds Inhibiting digOXin binding showed remarkable cOntractile effect In left etrlum end aorta. One eppeared to be a competitive Inhibitor of digitalis compounds with Klof 15.1I1M, whereas another one was a non-compelltive Inhibitor. These findings may support the proposed steroidal structure for EDLF and prompt us to persist In the synthetic approach to EDLF. . -, .. _. -_. endogenous dlgitsiis-like factor, synthetic steroid derivatives, dlgltslls receptor, human placenta.
K3
K4
CIRCULATING ENDOGENOUS DIGITALIS-LIKE FACTOR(S) IN ESSENTIAL HYPERTENSION, A..fag, GP Bemlnl, P Del Bene, A Moretti, G Nicolini, A Salvetti. Istltuto Flslologla Clinica CNR and Clinlca Medica I, University of Plsa, Pisa, Italy. The role of endogenous digitalis-like factor(s) (EDLF) In essential hypertension has been postulated for some time, but stili now It Is controversial whether elevated concentrations of this factor(s) are present In plasma of hypertensive patients. On the assumption that It is an absolute requirement that EDLF binds to digitalis receptors on sodium pump, we developed a simple, specific and sensitive radioreceptor assay (RRA) to detect EDLF in sample extracts using human placenta membranes as receptors, 12SI-dlgoxln as tracer, and ouabain as calibrator (Clin Chem, In press). EDLF values were hence expressed as ouabain-equivalents. Before RRA, plasma samples were extracted through disposable C18·cartrldges by 25% acetonitrile followed by pure acetonitrile to separate EDLFs with polarity similar, respectively, to ouabain (EDLF-1) and digoxin (EDLF-2). Here we report preliminary data on cirCUlating EDLF levels In essential hypertensive patients and In healthy controls. Two plasma samples from 22 untreated, essential hypertensive patients (11 males, 12 females; age 23-70 ys; SSP 160.0±3.8 (mean±SEM); DSP 100.6±2.1) and 15 sex and age matched healthy subjects (SSP 138.0±4.5; DSP 87.1±2.8) were extracted within 24 hrs from blood collection and assayed by RRA. A large Interlndividual variability was observed in both groups. Plasma EDLF-l In healthy controls ranged from 0 to 438 pM, with a mean of 121±43 pM. In hypertensive patients, mean EDLF-l was elevated, although not significantly, at 250±50 pM. The concentration ranged from 0 to 772 pM, with 4 patients Showing values higher than mean EDLF·1 In healthy subjects+2SD. Plasma EDLF-2 In hypertensives was 953±366 pM (range 0-6773 pM), significantly higher than healthy controls (14±6 pM, range 0-60 pM, p<0.05). Key Words: endogenous digitalis-like 'actor, hypertension digitalis receptor, radioreceptorassay, placenta:
HYPERTENSIVE EFFECT OF IMMUNOSUPRESSIVE THERAPY wtm STEROIDS IN PATIENTS wtm MYOCARDITIS ~ D Bene, D Zivlwv Saponja, D I'lIClwvic, J Milin, R.lung, J
I'asi(jevic. D Stoj.IC.lnstitute ofCardioavascular Disease.Sremska Kamenica, Yugoslavia.
At our Institute 45 patients(PTS) with pathohistologically (PH) proven myocarditis (MYOl classified according to the Dallas Criteria (DC) undervent immunosupressive therapy (ISm), which consisted of steroide. for all PTS. The ISTH went on through six months, and hypertension as one of the steroide' s side effects, was expected. The aim ofthe study was to follow up the effects of the tSTH on the myocardium and to check the PH changes with rebiopsy (REEMB). Ati PTS were classified into subgrops after PH fmding' 23 PTS with active MYO, and 18 PTS with borderline MYO. After REEMB, PTS showed PH changes after six months therapy: 12 PTS were ongoing, 15 PTS healing and 18 PTS were heald MYO. The doses of Prednisolon for PTS with ac1ive MYO were 2mglkgl BW at the beginning of the first month of the ISm, for PTS with borderline MYO doses were lower-t mglkgBW. The last four months they were treated with maintenance dose of Prednisolon 02 mglkgIBW. Despite of our expectation the increase of bind pressure was not significant neither after the fIlS! month therapy nor after the end ofthe sixth month.Only slightly mcrease' but statlsllcally not significant p<0.05, in diastolic blood pressure was seen in PTS with active MYO.
Key Words:
myocarditis. endomyocardial biopsy, Immunosuppressive therapy.
POSTERS: Steroids
Kl ALDOSTERONE AND DAY- AND NIGHT TIME AMBULATORY BLOOD PRESSURE PROFILE. H verlooye, D Duprez-, M De Buyzere, and DL Clement-. University Hospital, Gent, Belgium. Both renin and angiotensio II playa key role in blood pressure (BP) regulation during postural changes. As aldosterone (ALD) release is in part determined by other factors, there might be debate whether subsequently ALD levels are related to usual BP. We studied day (09.00 am - 09.00 pm) and night time (09.00 pm - 09.00 am) BP profile in relaton to supine (representative for nIght time) and standing (representative for day time) ALD concentrations in 30 untreated patients (15 men, 15 women, age 47 ± 13 years) with normal or mildly increased ambulatory BP. Ambulatory BP (BP) recordings at 30 min intervals (SpaceLabs 90207) were analysed for day- and night time systolic (SBP), diastolic (OBP) and mean arterial BP (MAP). Table summarizes correlation coefficients (p values) between ABP data and ALD, adjusted for age and BM!. BP ALD (supine) ALD (standing) (mm Hg) (l1.5±7.5 ngldl) (20.9±1l.l ngldl) Day SBP 148±11 0.39 (0.03) 0.50 (0.007) Night SBP 130±14 0.58 (0.001) 0.64 (0.0003) Day MAP 110±9 0.46 (0.01) 0.40 (0.03) Night MAP 96±11 0.60 (0.0009) 0.60 (0.0008) Day OBP 93±9 0.37 (0.05) 0.25 (0.14) Night OBP 78±1l 0.55 (0.003) 0.51 (0.0006) Aldosterone concentrations in supine and standing positions are well correlated with baseline night-time ABP and to a lesser extent with daytime ABP.
Key Words: aldosterone, ambulatory blood pressure, postural changes
K2 SYNTHETIC STEROID DERIVATIVES AS CANDIDATES FOR ENDOGENOUS DIGITALIS·L1KE FACTORS: INTERACTION WITH HUMAN PLACENTA DIGITALIS RECEPTORS. ~ M Sakakibara, P Del Bene, A Uchida. CNR Institute of Clinical Physiology, Plsa, Italy, and Pharm Res Lab Kirln Brewery Co, Gunma, Japan. Allempts to Isolste and chsrscterlze the endogenous digitalis-like factor(s) (EDLF) In mammals have been limited by the small amount of substance typically Isolsted. DigOXin, ouabain, ouabain-like substances, and an ouabain Isomer have been reported as EDLF. On the basis of the molecular weight of Inagaml-Tamura's EDLF and reported EDLF properties, some of us hypothesized, as a possible structure for EDLF, a steroidal skeleton containing a 2,3·catechol structure lacking the lactonlc ring at the 17-posltion of genlns. Several oxygenated estrogen derivatives were hence synthesized and analysed for contractile effect in Isolated rat aorta and guinea pig left atnum, and also many digitoxin dsrlvatives. Aim of this study was to analyse the reactivity of these synthetic compounds to digitalis receptors from human placenta. Using 1251-digoxln as tracer, receptor Interaction was analysed at equilibrium end by a sequential ssturation technique that we found to Incresse the sensitivity of the radioreceptor assay for EDLF detection In plasma. The question whether synthetic compounds Inhibiting digoxin binding acted via the dlgllalls binding site was enalysed by measuring 3H· ouabain binding to placental membranas at various, fixed concenlratlons of the Inhibllor. We found that specific 1251-digoxln binding to placental receptors wss Inhibited by 3 of the 14 compounds tested with ICso ranging between 10 and 10011M. Under the same conditions, ouabain and digoxin were effective at 5.9nM and B.4nM, digitoxigenin and dlgoxlgenln at 30nM, progesterone at 1011M, DHEA·S, cortisol and estradiol st >lOOI1M. Two of 3 synthetic compounds Inhibiting digOXin binding showed remarkable cOntractile effect In left etrlum end aorta. One eppeared to be a competitive Inhibitor of digitalis compounds with Klof 15.1I1M, whereas another one was a non-compelltive Inhibitor. These findings may support the proposed steroidal structure for EDLF and prompt us to persist In the synthetic approach to EDLF. . -, .. _. -_. endogenous dlgitsiis-like factor, synthetic steroid derivatives, dlgltslls receptor, human placenta.
K3
K4
CIRCULATING ENDOGENOUS DIGITALIS-LIKE FACTOR(S) IN ESSENTIAL HYPERTENSION, A..fag, GP Bemlnl, P Del Bene, A Moretti, G Nicolini, A Salvetti. Istltuto Flslologla Clinica CNR and Clinlca Medica I, University of Plsa, Pisa, Italy. The role of endogenous digitalis-like factor(s) (EDLF) In essential hypertension has been postulated for some time, but stili now It Is controversial whether elevated concentrations of this factor(s) are present In plasma of hypertensive patients. On the assumption that It is an absolute requirement that EDLF binds to digitalis receptors on sodium pump, we developed a simple, specific and sensitive radioreceptor assay (RRA) to detect EDLF in sample extracts using human placenta membranes as receptors, 12SI-dlgoxln as tracer, and ouabain as calibrator (Clin Chem, In press). EDLF values were hence expressed as ouabain-equivalents. Before RRA, plasma samples were extracted through disposable C18·cartrldges by 25% acetonitrile followed by pure acetonitrile to separate EDLFs with polarity similar, respectively, to ouabain (EDLF-1) and digoxin (EDLF-2). Here we report preliminary data on cirCUlating EDLF levels In essential hypertensive patients and In healthy controls. Two plasma samples from 22 untreated, essential hypertensive patients (11 males, 12 females; age 23-70 ys; SSP 160.0±3.8 (mean±SEM); DSP 100.6±2.1) and 15 sex and age matched healthy subjects (SSP 138.0±4.5; DSP 87.1±2.8) were extracted within 24 hrs from blood collection and assayed by RRA. A large Interlndividual variability was observed in both groups. Plasma EDLF-l In healthy controls ranged from 0 to 438 pM, with a mean of 121±43 pM. In hypertensive patients, mean EDLF-l was elevated, although not significantly, at 250±50 pM. The concentration ranged from 0 to 772 pM, with 4 patients Showing values higher than mean EDLF·1 In healthy subjects+2SD. Plasma EDLF-2 In hypertensives was 953±366 pM (range 0-6773 pM), significantly higher than healthy controls (14±6 pM, range 0-60 pM, p<0.05). Key Words: endogenous digitalis-like 'actor, hypertension digitalis receptor, radioreceptorassay, placenta:
HYPERTENSIVE EFFECT OF IMMUNOSUPRESSIVE THERAPY wtm STEROIDS IN PATIENTS wtm MYOCARDITIS ~ D Bene, D Zivlwv Saponja, D I'lIClwvic, J Milin, R.lung, J
I'asi(jevic. D Stoj.IC.lnstitute ofCardioavascular Disease.Sremska Kamenica, Yugoslavia.
At our Institute 45 patients(PTS) with pathohistologically (PH) proven myocarditis (MYOl classified according to the Dallas Criteria (DC) undervent immunosupressive therapy (ISm), which consisted of steroide. for all PTS. The ISTH went on through six months, and hypertension as one of the steroide' s side effects, was expected. The aim ofthe study was to follow up the effects of the tSTH on the myocardium and to check the PH changes with rebiopsy (REEMB). Ati PTS were classified into subgrops after PH fmding' 23 PTS with active MYO, and 18 PTS with borderline MYO. After REEMB, PTS showed PH changes after six months therapy: 12 PTS were ongoing, 15 PTS healing and 18 PTS were heald MYO. The doses of Prednisolon for PTS with ac1ive MYO were 2mglkgl BW at the beginning of the first month of the ISm, for PTS with borderline MYO doses were lower-t mglkgBW. The last four months they were treated with maintenance dose of Prednisolon 02 mglkgIBW. Despite of our expectation the increase of bind pressure was not significant neither after the fIlS! month therapy nor after the end ofthe sixth month.Only slightly mcrease' but statlsllcally not significant p<0.05, in diastolic blood pressure was seen in PTS with active MYO.
Key Words:
myocarditis. endomyocardial biopsy, Immunosuppressive therapy.