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An association study between essential hypertension (EH) and the dopamine D3 receptor gene (DRD3)
AJR-APRIL 1995-VOL.8, NO.4, PART 2
POSTERS: Geneticsand Molecular Biology of Hypertension 49A
AS
A6
EFFECT OF M235T GENOTYPE ON ANGJOTENSINOGEN tEVELS IN 25PAIRS OF TIVINS. M.P, Rutkowski, Y.B. Su, ItA. Tewksbury", V. Carter", P.S. Gartside, e. Klanke, A. Menon, M,e. ReW, University of Cincinnati College of Medicine, Cincinnati, OH, and Marshfield Medical Research Foundation, Marshfield, WI. We studied the relationship between anglotenslnogen (AGT) levels and the M235T AGT genotype In 25 pairsof twins (17 monozygotic, 8 dizygotic). 21 pairs were concordant for hypertension, 4 discordant. Of the 50 individuals, 33 were female and 4 were black. Mean agewas56. Plasma AGT was assayed by exhaustive digestion with renin followed by RIA. The Met235 (M) ~ Thr (T) mutation wasdetected by PCR (Human Molec Genet 1993;2:609-610). Log transformations of ACT levels and of AGT intrapair differences wereused as dependent variabtes in the general linear model procedure ofSAS. n Genotype Allele frequency
SAlT·INDUCED RENIN·UKE mRNA IN DAHL RATS S,M, WHnA-, E.W. Manhelmer .nd D.N. D.rllngton. Unlver.'ty
MM
Mf
IT
T
All 50 20 24 6 0,36 Hypertensive 46 18 22 6 0,37 ACT (ng AI/mil 40· 1663 2084 1740 (·=noestrogen) Multivariate analysis of the entiregroup demonstrated a significant effect of genotype (p=,022), gender (p=,OOl), body mass index (p=,OOOt) and estrogen (p=./J06) on AGT level. AGT Intrapair differences were not significantly different between the monozygotlc and dizygotic groups, Multivariate analysis for AGT intrapair differences in the monozygotic twins demonstrated no significant effect of genotype, age, gender, body mass index, estrogen or ACE inhibitor on ACT levels. Of note, in one monozygotic pair discordant {orhypertension, a high ACT level (3792 vs. 2065) induced ~y·~trogen didnotresult inhypertenstcn. Wecon\':lude that the M235T mutation is associated with modest increases In AGT levels. In the monozygotic subset we were unable to showa geneeffect on ACT level Or variability. Key Words:
essential hypertension, angiotensinogen, twintl, genetics, human.
A7 EVALUATION OF THE FUNCTIONAL IMPORTANCE OF TISSUE PRORENIN USING TRANSGENIC MICE. I.L. Reudelbubert, D. Methot and D. Silversides. MRC Group on Hypertension, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. The expression of the renin gene in extrarenal tissues (including the adrenal and pituitary glands and reproductive tissues) has led many investigators to propose the existence of locally active tissue renin-angiotensin systems (RAS). Although current evidence indicates that extrarenal tissues secrete only the inactive precursor of renin, prorenin, it has been !:l~8gested that prorenin could beactivated within tissues by a confonnauons! change which leads to unmasking of the enzyme active site, We have tested this hypothesis by selectively expressing either human active renin, human prorenin or human angiotensinogen in the anterior pituitary of transgenic mice. Our results demonstrate that expression of human active renin gives rise to an increased frequency of male sterility and gonadal atrophy in several transgenic mouse lines. Transgenic mice expressing both active human renin andhuman angictensinogen exhibit growth retardation. Notably, expression of human prorenin either alone or in combination with angiotensinogen has no discernible effect on either reproductive capacity or growth of three lines of transgenic mice tested. These results strongly suggest that human prorenin is not activated within tissues by a continuation-induced unmasking of the active site. They also suggest that if extrarenal RAS are funaionally important, they would either need to express (transiently?) enzymes capable of activating prorenin by proteolysis or to take up active renin from thecirculation, Key Words:
renin, prorenin, transgenic mice
of Maryland at Baltimore Sellool of Medicine, Baltimore, MD. Studies have shown thlt there ere structural differences betweon the Dahl hlt·sensitive lSI and Dahl lalt·r&sistant IRI renin genes and these rllnln alleles co·segregate with blood pressure. In the present study, we compared the responses of S and A renin gana expressIon to the changes In sodium Intake. We found that plasma renin activities of S rats were not suppressod after salt·loading. PRA Ing Allmllhrl PRe IngAllmllhr) Diet S R S R
8% NaCI O.49±0.13 0.26:1:0,03 1,89:1:0.57 0.oa:~·O.06 0.4% NaCI 0.86:t0.19 1.05:1:0.20 1.05:1:0.18 o.a::':tO,25 Na-deficient 7.81:1; 1.0110.75:1: 1.526.61:1: 1.21 17.56:1:2.87
When total renal RNA from these rata wero Isolated and analyzed by Northern blothybridization using 8 Sprague·Dawley rat renin eDNA as probe, we found that the expression of renin mRNA 11 .6 kb) wassuppressed bythe Increase of sodium Intake In both stralnc of Dahl rats. However, an additio:lal band 11.9 kb) with less intensity was alsodetected and its expressiclO was enhanced by salt·loading alld suppresaed by sodium depletion. The lavel of the 1.9 kb renin·like mR~lA of tt.e 5 rat is much higher than that of the R rat, It Is highly posslbie that the gene product of this salt·lnduced renln- like mRNA might accountfor the unsuppressed plasma renin activity seen In the S rats end contributes to the pathogenasJs of the ult·hypertensJve effect seen In the S rat. Wehed Isolated and clonod the 5 and R 1.9 kb lonin'like mRNAs from rats maintained On high salt·diet. The functJonal significance of pOlis/bIB protein productof the 1.9 kb renin·like mRNA In renin·anglotensln system Is currentlv under investigation.
Key Words: renin-angiotensin system, genetic hypertension, renin gene expreS6ion
AS AN ASSOCIATION STUDY BETWEEN ESSENTIAL HYPERTENSION (EH) AND TIlE OOPAMINE 03 RECEPTOR GENE (DRD3). O.M'] NAIMABK. M MASEllIS, F. BADRI, N, KING, T. KLEMPAN, H. WINTER, A PETRGNIS, T,PARSONS, JL. KENNEDY. Clarke Institute, University ofToronto. Toronto ON. ClIlIada.
Dopamine is IIlI important regulator of intra·renal hemodynamics IIlId tubular sodium reabsorption. Defects in dopamine re.:eptor structure orfunctioo may rcsuIt inimpaired excretion of sodiu.'Il and contribute to the development of EH. The Dopamine D3 receptor msyplay 8 role inintra-renal sodium r.mdling since itsmRNA hasbeen recently lIemonstrated byGao et a/,· inglomeruli. proximaltubuJcs and microvessels from WKY and SHR ki~.ey. Accordi"gly. esa preliminary investigation in humans, we have conducted an ASSOCiation study comparing the distributions of a restriction fragment length polymorphism of the OR03 gene between patients with essential hypertension (on at least one antihypertensive medication) and normotensive oootrols. After obtaining informed consent, venous blood samples were obtained and DNA was eldraCted non·enzymatically from peripheralleukocytcs. Using the polymerase chain reaction, 1\304bp fragment from exon I ofOR03 was amplified and then subj~ted to restriction endonuclease digestion with MscI. The digested products were then subjected toagarose-gel electrophoresis and visualized with ethidium bromide. Presencl: orabsence oftherestriction sitedelineated IWt'ORO) alleles denoted I and 2. Amcmg 176controls thedistribution of 1.1, 1-2 and 2·2 genotypes was 80. 73 and 23 individuals respectively mille the corresponding distribution in 17patients with EH was 7,7. and 3. In the controls thefrequenciesfor alleles I and2 were 0.662 and 0.338 ~ely \\iille thecorresponding frequencies in the patients with ElI were 0.618 and 0.382 respectively (X2 =0.11, p = 0.13). The results ofthis prelirninnry investigation donotsupport an associationbetween theDRD3 genetic locus and essential hypertension. More extensive genolyping ofpatients with EH will be necessary to confl!Ill these findings. *Gao DQ., Am. J.Physiol. 1994, 266(4p(2):F646-S0. Key Words:
h i Dopamine, DRD3 , po1ymerase can reaction, RFLP
POSTERS: Geneticsand Molecular Biology of Hypertension 49A
AS
A6
EFFECT OF M235T GENOTYPE ON ANGJOTENSINOGEN tEVELS IN 25PAIRS OF TIVINS. M.P, Rutkowski, Y.B. Su, ItA. Tewksbury", V. Carter", P.S. Gartside, e. Klanke, A. Menon, M,e. ReW, University of Cincinnati College of Medicine, Cincinnati, OH, and Marshfield Medical Research Foundation, Marshfield, WI. We studied the relationship between anglotenslnogen (AGT) levels and the M235T AGT genotype In 25 pairsof twins (17 monozygotic, 8 dizygotic). 21 pairs were concordant for hypertension, 4 discordant. Of the 50 individuals, 33 were female and 4 were black. Mean agewas56. Plasma AGT was assayed by exhaustive digestion with renin followed by RIA. The Met235 (M) ~ Thr (T) mutation wasdetected by PCR (Human Molec Genet 1993;2:609-610). Log transformations of ACT levels and of AGT intrapair differences wereused as dependent variabtes in the general linear model procedure ofSAS. n Genotype Allele frequency
SAlT·INDUCED RENIN·UKE mRNA IN DAHL RATS S,M, WHnA-, E.W. Manhelmer .nd D.N. D.rllngton. Unlver.'ty
MM
Mf
IT
T
All 50 20 24 6 0,36 Hypertensive 46 18 22 6 0,37 ACT (ng AI/mil 40· 1663 2084 1740 (·=noestrogen) Multivariate analysis of the entiregroup demonstrated a significant effect of genotype (p=,022), gender (p=,OOl), body mass index (p=,OOOt) and estrogen (p=./J06) on AGT level. AGT Intrapair differences were not significantly different between the monozygotlc and dizygotic groups, Multivariate analysis for AGT intrapair differences in the monozygotic twins demonstrated no significant effect of genotype, age, gender, body mass index, estrogen or ACE inhibitor on ACT levels. Of note, in one monozygotic pair discordant {orhypertension, a high ACT level (3792 vs. 2065) induced ~y·~trogen didnotresult inhypertenstcn. Wecon\':lude that the M235T mutation is associated with modest increases In AGT levels. In the monozygotic subset we were unable to showa geneeffect on ACT level Or variability. Key Words:
essential hypertension, angiotensinogen, twintl, genetics, human.
A7 EVALUATION OF THE FUNCTIONAL IMPORTANCE OF TISSUE PRORENIN USING TRANSGENIC MICE. I.L. Reudelbubert, D. Methot and D. Silversides. MRC Group on Hypertension, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. The expression of the renin gene in extrarenal tissues (including the adrenal and pituitary glands and reproductive tissues) has led many investigators to propose the existence of locally active tissue renin-angiotensin systems (RAS). Although current evidence indicates that extrarenal tissues secrete only the inactive precursor of renin, prorenin, it has been !:l~8gested that prorenin could beactivated within tissues by a confonnauons! change which leads to unmasking of the enzyme active site, We have tested this hypothesis by selectively expressing either human active renin, human prorenin or human angiotensinogen in the anterior pituitary of transgenic mice. Our results demonstrate that expression of human active renin gives rise to an increased frequency of male sterility and gonadal atrophy in several transgenic mouse lines. Transgenic mice expressing both active human renin andhuman angictensinogen exhibit growth retardation. Notably, expression of human prorenin either alone or in combination with angiotensinogen has no discernible effect on either reproductive capacity or growth of three lines of transgenic mice tested. These results strongly suggest that human prorenin is not activated within tissues by a continuation-induced unmasking of the active site. They also suggest that if extrarenal RAS are funaionally important, they would either need to express (transiently?) enzymes capable of activating prorenin by proteolysis or to take up active renin from thecirculation, Key Words:
renin, prorenin, transgenic mice
of Maryland at Baltimore Sellool of Medicine, Baltimore, MD. Studies have shown thlt there ere structural differences betweon the Dahl hlt·sensitive lSI and Dahl lalt·r&sistant IRI renin genes and these rllnln alleles co·segregate with blood pressure. In the present study, we compared the responses of S and A renin gana expressIon to the changes In sodium Intake. We found that plasma renin activities of S rats were not suppressod after salt·loading. PRA Ing Allmllhrl PRe IngAllmllhr) Diet S R S R
8% NaCI O.49±0.13 0.26:1:0,03 1,89:1:0.57 0.oa:~·O.06 0.4% NaCI 0.86:t0.19 1.05:1:0.20 1.05:1:0.18 o.a::':tO,25 Na-deficient 7.81:1; 1.0110.75:1: 1.526.61:1: 1.21 17.56:1:2.87
When total renal RNA from these rata wero Isolated and analyzed by Northern blothybridization using 8 Sprague·Dawley rat renin eDNA as probe, we found that the expression of renin mRNA 11 .6 kb) wassuppressed bythe Increase of sodium Intake In both stralnc of Dahl rats. However, an additio:lal band 11.9 kb) with less intensity was alsodetected and its expressiclO was enhanced by salt·loading alld suppresaed by sodium depletion. The lavel of the 1.9 kb renin·like mR~lA of tt.e 5 rat is much higher than that of the R rat, It Is highly posslbie that the gene product of this salt·lnduced renln- like mRNA might accountfor the unsuppressed plasma renin activity seen In the S rats end contributes to the pathogenasJs of the ult·hypertensJve effect seen In the S rat. Wehed Isolated and clonod the 5 and R 1.9 kb lonin'like mRNAs from rats maintained On high salt·diet. The functJonal significance of pOlis/bIB protein productof the 1.9 kb renin·like mRNA In renin·anglotensln system Is currentlv under investigation.
Key Words: renin-angiotensin system, genetic hypertension, renin gene expreS6ion
AS AN ASSOCIATION STUDY BETWEEN ESSENTIAL HYPERTENSION (EH) AND TIlE OOPAMINE 03 RECEPTOR GENE (DRD3). O.M'] NAIMABK. M MASEllIS, F. BADRI, N, KING, T. KLEMPAN, H. WINTER, A PETRGNIS, T,PARSONS, JL. KENNEDY. Clarke Institute, University ofToronto. Toronto ON. ClIlIada.
Dopamine is IIlI important regulator of intra·renal hemodynamics IIlId tubular sodium reabsorption. Defects in dopamine re.:eptor structure orfunctioo may rcsuIt inimpaired excretion of sodiu.'Il and contribute to the development of EH. The Dopamine D3 receptor msyplay 8 role inintra-renal sodium r.mdling since itsmRNA hasbeen recently lIemonstrated byGao et a/,· inglomeruli. proximaltubuJcs and microvessels from WKY and SHR ki~.ey. Accordi"gly. esa preliminary investigation in humans, we have conducted an ASSOCiation study comparing the distributions of a restriction fragment length polymorphism of the OR03 gene between patients with essential hypertension (on at least one antihypertensive medication) and normotensive oootrols. After obtaining informed consent, venous blood samples were obtained and DNA was eldraCted non·enzymatically from peripheralleukocytcs. Using the polymerase chain reaction, 1\304bp fragment from exon I ofOR03 was amplified and then subj~ted to restriction endonuclease digestion with MscI. The digested products were then subjected toagarose-gel electrophoresis and visualized with ethidium bromide. Presencl: orabsence oftherestriction sitedelineated IWt'ORO) alleles denoted I and 2. Amcmg 176controls thedistribution of 1.1, 1-2 and 2·2 genotypes was 80. 73 and 23 individuals respectively mille the corresponding distribution in 17patients with EH was 7,7. and 3. In the controls thefrequenciesfor alleles I and2 were 0.662 and 0.338 ~ely \\iille thecorresponding frequencies in the patients with ElI were 0.618 and 0.382 respectively (X2 =0.11, p = 0.13). The results ofthis prelirninnry investigation donotsupport an associationbetween theDRD3 genetic locus and essential hypertension. More extensive genolyping ofpatients with EH will be necessary to confl!Ill these findings. *Gao DQ., Am. J.Physiol. 1994, 266(4p(2):F646-S0. Key Words:
h i Dopamine, DRD3 , po1ymerase can reaction, RFLP