ABSTRACT
BACKGROUND AND PURPOSE: For many years, it was suspected that sheep expressed only one melatonin receptor (closely resembling MT(1) from other mammal species). Here we report the cloning of another melatonin receptor, MT(2), from sheep. EXPERIMENTAL APPROACH: Using a thermo-resistant reverse transcriptase and polymerase chain reaction primer set homologous to the bovine MT(2) mRNA sequence, we have cloned and characterized MT(2) receptors from sheep retina. KEY RESULTS: The ovine MT(2) receptor presents 96%, 72% and 67% identity with cattle, human and rat respectively. This MT(2) receptor stably expressed in CHO-K1 cells showed high-affinity 2[(125)I]-iodomelatonin binding (K(D)= 0.04 nM). The rank order of inhibition of 2[(125)I]-iodomelatonin binding by melatonin, 4-phenyl-2-propionamidotetralin and luzindole was similar to that exhibited by MT(2) receptors of other species (melatonin > 4-phenyl-2-propionamidotetralin > luzindole). However, its pharmacological profile was closer to that of rat, rather than human MT(2) receptors. Functionally, the ovine MT(2) receptors were coupled to G(i) proteins leading to inhibition of adenylyl cyclase, as the other melatonin receptors. In sheep brain, MT(2) mRNA was expressed in pars tuberalis, choroid plexus and retina, and moderately in mammillary bodies. Real-time polymerase chain reaction showed that in sheep pars tuberalis, premammillary hypothalamus and mammillary bodies, the temporal pattern of expression of MT(1) and MT(2) mRNA was not parallel in the three tissues. CONCLUSION AND IMPLICATIONS: Co-expression of MT(1) and MT(2) receptors in all analysed sheep brain tissues suggests that MT(2) receptors may participate in melatonin regulation of seasonal anovulatory activity in ewes by modulating MT(1) receptor action.
Subject(s)
Receptor, Melatonin, MT2/genetics , Amino Acid Sequence , Animals , Brain/metabolism , CHO Cells , Cattle , Cloning, Molecular , Cricetinae , Cricetulus , Female , GTP-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Organ Specificity , RNA, Messenger/metabolism , Radioligand Assay , Rats , Receptor, Melatonin, MT1/antagonists & inhibitors , Receptor, Melatonin, MT1/metabolism , Receptor, Melatonin, MT2/antagonists & inhibitors , Receptor, Melatonin, MT2/metabolism , Recombinant Proteins/metabolism , Retina/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sheep , Tetrahydronaphthalenes/pharmacology , Tryptamines/pharmacologyABSTRACT
Anomalies in either of the tightly linked genes encoding the enzymes CYP11B1 (11beta-hydroxylase) or CYP11B2 (aldosterone synthase) can lead to important changes in arterial pressure and are responsible for several monogenically inherited forms of hypertension. Mutations in these genes or their regulatory regions could thus contribute to genetic variation in susceptibility to essential hypertension. To test this hypothesis, we performed 2 complementary studies of the CYP11B1/CYP11B2 locus in essential hypertension. After characterizing a DNA contig containing the CYP11B1 gene and mapping the gene in the Centre d'Etudes du Polymorphisme Humain reference panel of families, we performed a linkage study with 292 hypertensive sibling pairs and a highly informative microsatellite marker near CYP11B1. We also analyzed the association of 2 frequent biallelic polymorphisms of the CYP11B2 gene, 1 in the promoter at position -344 (-344C/T) and the other, a common gene conversion in intron 2, with hypertension in 380 hypertensive patients and 293 normotensive individuals. Statistical analyses did not show significant linkage of the CYP11B1 microsatellite marker to hypertension. No positive association with hypertension was found with the gene conversion in intron 2, but a positive association with hypertension was found with the -344T allele. The hypertensive and normotensive samples differed significantly in both genotype (P=0.023) and allele frequencies (P=0.010). Our data suggest a modest contribution of the CYP11B2 gene to essential hypertension.
Subject(s)
Cytochrome P-450 CYP11B2/genetics , Hypertension/genetics , Mutation , Adult , Female , Gene Frequency , Genetic Linkage , Humans , Male , Middle Aged , Polymorphism, GeneticABSTRACT
We conducted the present study to determine whether the angiotensin II type I receptor (AT1) gene might be implicated in human essential hypertension by using case-control and linkage studies. The entire coding and 3' untranslated regions of the AT1 receptor gene (2.2 kb) were amplified by polymerase chain reaction and submitted to single-strand conformation polymorphism in 60 hypertensive subjects with a familial susceptibility. We identified five polymorphisms (T573-->C, A1062-->G, A1166-->C, G1517-->T, and A1878-->G). However, no mutations that alter the encoded amino acid sequence were detected. A case-control study performed on white hypertensive (n = 206; blood pressure, 168 +/- 16/103 +/- 9 mm Hg) and normotensive (n = 298; blood pressure, 122 +/- 10/75 +/- 9 mm Hg) subjects using three of five polymorphisms showed a significant increase in allelic frequency of C1166 in hypertensive subjects (0.36 versus 0.28 for normotensive subjects, chi 2 = 6.8, P < .01). Frequencies for the alleles of the other two polymorphisms (T573-->C, A1878-->G) were similar in both groups. We performed a linkage study using the affected sib pair method and a highly polymorphic marker of the AT1 receptor gene. There was no evidence for linkage in 267 sib pairs analyzed from 138 pedigrees. These findings would be compatible with a common variant of the AT1 receptor imparting a small effect on blood pressure; further studies will be needed to address this possibility.
