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Polymorphisms of the γ subunit of the epithelial sodium channel in essential hypertension. Relationship with salt-sensitivity
AJH-APRIL
1999-VOL.
12, NO. 4, PART 2
ORALS:
Theme
III: Are the Results
of Clinical
Trials
Us&I
in Practice?
A001
A002
IN WV0 GENE-TRANSFER OF CALCITONIN GENE-RELATED PEPTIDE (CGRP) REDUCES HYPOXIA-lNDUCED PULMONARY HYPERTENSION IN THE MOUSE. mChamrrion, TJ Bivalacqw
IDENTIFICATION OF MOLECULAR DETERMINANTS OF ATRIAL NATRIURETIC PEF’TIDE RECEPTOR - A BY DELETION MUTAGENESIS. m$ R Kumar, K Pandey, W Cartledge, and M Li, Dept. of Physiology. Tulane University School of Medicine, New Orleans, LA, and Medical College of Georgia, Augusta, GA. Objective of this study was to determine the functional domains of atria1 natriuretic peptide receptor-A (Npra) in post-binding events and metabolic turnover of ligand-receptor complexes. Using sequential deletion mutagenesis, our studies have determined the specific regions in the intracellular domains of Npra, relevant to receptor function during the biological action of ANP. The plasmid pGEMSZ(+) was used to delete the Npra cDNA in 3-5 orientation. The deleted cDNA inserts were removed from plasmid of pGEMSZ(+) and ligated into the mammalian expression vector pcDNA3, modified to contain TGA stop codon. The wild-type and sequentially deleted Npra cDNA inserts were transiently transfected into COS-7 cells by electroporation. Transfected cells were plated in 6Omm dishes and cultured at 37oC in an atmosphere of 5% CO2 and 95% Oz. After 48 h, functional studies were carried out using either wild-type or mutant Npra. The deletion of 13 to 120 amino acid residues at the carboxyl-terminal end of Npra diminished the ligand binding, cGMP production and internalization of ligand-receptor complexes by 45 85 % and 90%, respectively. The results demonstrate that specific sequence motifs in the intracellular domain of Npra determine the extent of ANP-binding, metabolic turnover and receptor sequestration in transfected COS-7 cells.
F.M. D’Souza, Kazunori Toyoda, DD Heistad, A.L. Hyman’ and PJ Kadowitz, Tulane University School of Medicine, New Orleans, LA. Since pulmonary hypertension (PH) is still a condition with a very war txognosis, the delivery of genes for vasodilator peptides may prove The purpose of the io bC u&l in the treat&t %f this condition. mesent studv was to investieate the et?bd of eene-transfer of CGRP, a ‘wtent pulmbnary vasodilato~, on PH and rigit ventricular hypertmphy iRVH;‘as meas&d by RV/(LV+S)] in res+se to chronic hypoxic exwsure 12 weeks: FIO, 0.10). A new rieht-heart catheterization technique was used to m&sure bulmonary a&al pressure (PAP) and pulmon~ arterial wedge pressure (PAWP) in the intact-chest anesthetized mouse and cardiac output (CO) was determined by the thermodilution technique. Five days before the mice were exposed to hypoxia, adenoviral RSV vector ‘containing the gene for CGRP (AdRSVCGRP) or the reporter gene for Pgalactosidase (AdRSVi3gal) was administered intratracheally. The increase in PAP, PVR, and RV/(LV+S) that was observed in vehicle-treated and AdRSVpgal-treated mice two weeks afler exposure to hypoxia was significantly reduced in mice treated with AdRSVCGRP. Systemic arterial pressure and total peripheral resistance were similar in all groups of mice. Pulmonary IV CAMP-selective vasodepressor responses to the type phosphodiesterase inhibitor, rolipram, were significantly enhanced in In addition, pulmonary animals treated with AdRSVCGRP. vasopressor responses to endothelin-I, angiotensin II. and the thmmboxane AZ mimic U46619 were sianiticantlr reduced in mice treated with AbRSVCGRP when comp&d to AdRSVpgal-treated animals while systemic responses to all agonists were not altered. RlA of whole lung tissue showed increased levels of CGRP and CAMP, but MOlZOV% not cGMP, in mice hansfected with AdRSVCGRP. histochemistry of lung slices showed viral transfection of the repotter gene in resistance-sized (IOO-300pm) pulmonary blood vessels. These data provide evidence that in viva gene-transfer of the potent vasodilator CGRP can attenuate the increases in PAP and PVR, and the RVH associated with chronic hypoxia without altering systemic vascular parameters. This is to our knowledge the first report of a gene-transfer technique being used to reduce the PH and RVH induced by chronic hypoxia and may provide insight into a novel treatment for PH. Key Words:
gene-transfer, mouse, pulmonary hypertension hypoxia, calcitonin-gene related peptide (CGRP)
%,
KeYWords:
atria1 natriuretic mutagenesis.
peptide,
receptor,
cDNA,
A003
A004
POLYMORPHISMS OF TIIE y SUBUNIT OF THE EPITRELIAL SODIUM CHANNEL IN ESSENTIAL WPERTENSION. RELATIONSHIP WITFI SALTSENSITIVITY E. Poch, V. Giner, D. Gonzalez, A. de la Sierra*, A. Botey, A. Coca*, A Dar&, F. Rivera. Departments ofNephroogy, Internal Medicine and Hormonology. Hospital Clinic. Barcelona. Spain. The aim of the present study was to evaluate the association of a common polymorphism in the y subunit of the epithelial sodium channel (yENaC), with essential hypertension and the blood pressure response to high salt intake. We studied 111 essential hypertensive patients (54 m and 57 f), aged 54*1 years and 112 nonnotensive controls (65 m and 43 f), aged 46+1 years. Salt-sensitivity, detined as a significant increase in 24-hour man blood pressure fiom low-salt (20 mmol/day) to high-salt (260 mmol/day) intake, was assessed in a subgroup of 30 hypertensive patients. The genotype distribution in essential hypertensive patients (66%CC and 34%CG/GG) was not significantly different from the normotensive control group (57%CC and 43%CG/GG; p=O.21). Age, gender distribution, office systolic and diastolic blood pressure, plasma renin activity and plasma aldosterone levels were not significantly different between essential hypertensives classified according to the yENaC genotype. Likewise, in 30 patients class&d accordiig to the saltsensitivity status, the genotype distribution did not differ between salt-sensitive patients (64%CC, 36%CG/GG) and salt-resistant hypertensives (74%CC, 26%CG/GG; p=O.68). These results do not support an association between yENaC polymorphism and either essential hypertension nor saltandsuggest thatmutations in EN& are unlikely to ly contribute to the pathogenesis of this disease, genetics, salt-sensitivity, sodium channel
ACE-I GENE F’OLYMORFHISM AND ARTERIAL HYPERTENSION: LACK OF ASSGCIATION IN AFRaVRNEZUELAN POPULATIONS AJ. Dehzad~, R. Palacio, R. Farlas, V. Mago, J. Cermetio, F. Fragach&n*. Unidad de Hipertension Arterial, Hospital Universitario de Caracas, Universidad Central de Venezuela The ACE-I gene l/D polymorphism is considered an independent risk factor for coronary heart disease, and despite is not associated with arterial hypertension (AH), the deletion allele @ allele) sets the stage for the atherosclerotic and renal microvascular complications of this disease. The aim of study was to determine the associations between l/D polymorphism with AH in a welldefined ethnic group. The participants came from Barlovento region (The Barlovento Study); those have 76790/0 of black genetic contribution. They were divided in 2 groups: Hypertensive (HT) and Nomtotensive (NT). l/D polymorphism was determined by PCR. Genotype‘s distribution: HT (t&2) DD 12, ID 7, II 3; NT (n=23) DD 11, ID 8, H 4; MaIe (n=31) DDl8, ID 10, II 3; FemaIe(n=l4) DD 5, ID 5, H 4. Allele’s distribution: HT D 0.705 I 0.295; NT D 0.50 I 0.50; Male D 0.742 I 0.258; Female D 0.536 I 0.464. No statistical difference were found in the distribution of the genotypes (ET vs. NT x2=0.231, p 0.891, MaIe vt~ Female x2=01.29, p 0.525) and the frequency of the alleles (HT vs. NT x2=0.412, p 0.521). Statistical difference in the frecuency of the alleles of Mak vs. Femak (x2=8.233. p 0.004), being D allele mom Sequent in Male. No significant associations were observed between ACE gene polymorphism and AH. AfroVenezuelan HT men may have an increased risk for atherosclerotic and renal microvascular complications.
43A
1999-VOL.
12, NO. 4, PART 2
ORALS:
Theme
III: Are the Results
of Clinical
Trials
Us&I
in Practice?
A001
A002
IN WV0 GENE-TRANSFER OF CALCITONIN GENE-RELATED PEPTIDE (CGRP) REDUCES HYPOXIA-lNDUCED PULMONARY HYPERTENSION IN THE MOUSE. mChamrrion, TJ Bivalacqw
IDENTIFICATION OF MOLECULAR DETERMINANTS OF ATRIAL NATRIURETIC PEF’TIDE RECEPTOR - A BY DELETION MUTAGENESIS. m$ R Kumar, K Pandey, W Cartledge, and M Li, Dept. of Physiology. Tulane University School of Medicine, New Orleans, LA, and Medical College of Georgia, Augusta, GA. Objective of this study was to determine the functional domains of atria1 natriuretic peptide receptor-A (Npra) in post-binding events and metabolic turnover of ligand-receptor complexes. Using sequential deletion mutagenesis, our studies have determined the specific regions in the intracellular domains of Npra, relevant to receptor function during the biological action of ANP. The plasmid pGEMSZ(+) was used to delete the Npra cDNA in 3-5 orientation. The deleted cDNA inserts were removed from plasmid of pGEMSZ(+) and ligated into the mammalian expression vector pcDNA3, modified to contain TGA stop codon. The wild-type and sequentially deleted Npra cDNA inserts were transiently transfected into COS-7 cells by electroporation. Transfected cells were plated in 6Omm dishes and cultured at 37oC in an atmosphere of 5% CO2 and 95% Oz. After 48 h, functional studies were carried out using either wild-type or mutant Npra. The deletion of 13 to 120 amino acid residues at the carboxyl-terminal end of Npra diminished the ligand binding, cGMP production and internalization of ligand-receptor complexes by 45 85 % and 90%, respectively. The results demonstrate that specific sequence motifs in the intracellular domain of Npra determine the extent of ANP-binding, metabolic turnover and receptor sequestration in transfected COS-7 cells.
F.M. D’Souza, Kazunori Toyoda, DD Heistad, A.L. Hyman’ and PJ Kadowitz, Tulane University School of Medicine, New Orleans, LA. Since pulmonary hypertension (PH) is still a condition with a very war txognosis, the delivery of genes for vasodilator peptides may prove The purpose of the io bC u&l in the treat&t %f this condition. mesent studv was to investieate the et?bd of eene-transfer of CGRP, a ‘wtent pulmbnary vasodilato~, on PH and rigit ventricular hypertmphy iRVH;‘as meas&d by RV/(LV+S)] in res+se to chronic hypoxic exwsure 12 weeks: FIO, 0.10). A new rieht-heart catheterization technique was used to m&sure bulmonary a&al pressure (PAP) and pulmon~ arterial wedge pressure (PAWP) in the intact-chest anesthetized mouse and cardiac output (CO) was determined by the thermodilution technique. Five days before the mice were exposed to hypoxia, adenoviral RSV vector ‘containing the gene for CGRP (AdRSVCGRP) or the reporter gene for Pgalactosidase (AdRSVi3gal) was administered intratracheally. The increase in PAP, PVR, and RV/(LV+S) that was observed in vehicle-treated and AdRSVpgal-treated mice two weeks afler exposure to hypoxia was significantly reduced in mice treated with AdRSVCGRP. Systemic arterial pressure and total peripheral resistance were similar in all groups of mice. Pulmonary IV CAMP-selective vasodepressor responses to the type phosphodiesterase inhibitor, rolipram, were significantly enhanced in In addition, pulmonary animals treated with AdRSVCGRP. vasopressor responses to endothelin-I, angiotensin II. and the thmmboxane AZ mimic U46619 were sianiticantlr reduced in mice treated with AbRSVCGRP when comp&d to AdRSVpgal-treated animals while systemic responses to all agonists were not altered. RlA of whole lung tissue showed increased levels of CGRP and CAMP, but MOlZOV% not cGMP, in mice hansfected with AdRSVCGRP. histochemistry of lung slices showed viral transfection of the repotter gene in resistance-sized (IOO-300pm) pulmonary blood vessels. These data provide evidence that in viva gene-transfer of the potent vasodilator CGRP can attenuate the increases in PAP and PVR, and the RVH associated with chronic hypoxia without altering systemic vascular parameters. This is to our knowledge the first report of a gene-transfer technique being used to reduce the PH and RVH induced by chronic hypoxia and may provide insight into a novel treatment for PH. Key Words:
gene-transfer, mouse, pulmonary hypertension hypoxia, calcitonin-gene related peptide (CGRP)
%,
KeYWords:
atria1 natriuretic mutagenesis.
peptide,
receptor,
cDNA,
A003
A004
POLYMORPHISMS OF TIIE y SUBUNIT OF THE EPITRELIAL SODIUM CHANNEL IN ESSENTIAL WPERTENSION. RELATIONSHIP WITFI SALTSENSITIVITY E. Poch, V. Giner, D. Gonzalez, A. de la Sierra*, A. Botey, A. Coca*, A Dar&, F. Rivera. Departments ofNephroogy, Internal Medicine and Hormonology. Hospital Clinic. Barcelona. Spain. The aim of the present study was to evaluate the association of a common polymorphism in the y subunit of the epithelial sodium channel (yENaC), with essential hypertension and the blood pressure response to high salt intake. We studied 111 essential hypertensive patients (54 m and 57 f), aged 54*1 years and 112 nonnotensive controls (65 m and 43 f), aged 46+1 years. Salt-sensitivity, detined as a significant increase in 24-hour man blood pressure fiom low-salt (20 mmol/day) to high-salt (260 mmol/day) intake, was assessed in a subgroup of 30 hypertensive patients. The genotype distribution in essential hypertensive patients (66%CC and 34%CG/GG) was not significantly different from the normotensive control group (57%CC and 43%CG/GG; p=O.21). Age, gender distribution, office systolic and diastolic blood pressure, plasma renin activity and plasma aldosterone levels were not significantly different between essential hypertensives classified according to the yENaC genotype. Likewise, in 30 patients class&d accordiig to the saltsensitivity status, the genotype distribution did not differ between salt-sensitive patients (64%CC, 36%CG/GG) and salt-resistant hypertensives (74%CC, 26%CG/GG; p=O.68). These results do not support an association between yENaC polymorphism and either essential hypertension nor saltandsuggest thatmutations in EN& are unlikely to ly contribute to the pathogenesis of this disease, genetics, salt-sensitivity, sodium channel
ACE-I GENE F’OLYMORFHISM AND ARTERIAL HYPERTENSION: LACK OF ASSGCIATION IN AFRaVRNEZUELAN POPULATIONS AJ. Dehzad~, R. Palacio, R. Farlas, V. Mago, J. Cermetio, F. Fragach&n*. Unidad de Hipertension Arterial, Hospital Universitario de Caracas, Universidad Central de Venezuela The ACE-I gene l/D polymorphism is considered an independent risk factor for coronary heart disease, and despite is not associated with arterial hypertension (AH), the deletion allele @ allele) sets the stage for the atherosclerotic and renal microvascular complications of this disease. The aim of study was to determine the associations between l/D polymorphism with AH in a welldefined ethnic group. The participants came from Barlovento region (The Barlovento Study); those have 76790/0 of black genetic contribution. They were divided in 2 groups: Hypertensive (HT) and Nomtotensive (NT). l/D polymorphism was determined by PCR. Genotype‘s distribution: HT (t&2) DD 12, ID 7, II 3; NT (n=23) DD 11, ID 8, H 4; MaIe (n=31) DDl8, ID 10, II 3; FemaIe(n=l4) DD 5, ID 5, H 4. Allele’s distribution: HT D 0.705 I 0.295; NT D 0.50 I 0.50; Male D 0.742 I 0.258; Female D 0.536 I 0.464. No statistical difference were found in the distribution of the genotypes (ET vs. NT x2=0.231, p 0.891, MaIe vt~ Female x2=01.29, p 0.525) and the frequency of the alleles (HT vs. NT x2=0.412, p 0.521). Statistical difference in the frecuency of the alleles of Mak vs. Femak (x2=8.233. p 0.004), being D allele mom Sequent in Male. No significant associations were observed between ACE gene polymorphism and AH. AfroVenezuelan HT men may have an increased risk for atherosclerotic and renal microvascular complications.
43A