Hisao Tanase

Phospholipase C-delta gene of the spontaneously hypertensive rat harbors point mutations causing amino acid substitutions in a catalytic domain.

This study was undertaken in order to investigate the newly discovered spontaneously hypertensive rat (SHR)-specific restriction fragment length polymorphism (RFLP) at the genomic locus of (poly)phosphoinositide-specific phospholipase C (PLC)-delta at a DNA sequence level. Our aim was to clone the PLC-delta complimentary DNA (cDNA) from SHR and analyse the genomic DNA obtained from two hypertensive rat strains such as SHR and its stroke-prone substrain (SHR-SP) and three normotensive rat strains such as Sprague-Dawley, Donryu and Wistar-Kyoto (WKY) by preparing an aortic cDNA library of SHR, hybridization cloning of PLC-delta cDNA and an analysis of the genomic DNA by polymerase chain reaction. By digesting with restriction enzyme XhoI, we discovered an RFLP band displaying only in SHR and SHR-SP, not in Sprague-Dawley, Donryu and WKY rats. DNA sequencing of PLC-delta cDNA cloned from an aortic cDNA library of SHR revealed a total of three SHR-specific point mutations, two of which resulted in amino acid substitutions. The first point mutation (A to T) was detected at the XhoI site, changing a threonine(ACG) to a serine(TCG), and the second point mutation (A to G) was discovered in the vicinity of the first one, changing an isoleucine(ATA) to a methionine(ATG). This is the first demonstration of the mutations in the SHR genome changing amino acid sequences. These amino acid substitutions, situated in the putative catalytic X domain of PLC-delta, may be the major cause of the augmented PLC activity observed in the SHR, possibly leading to hypertension-related phenonemoma such as abnormal calcium homeostasis and increased intracellular calcium ion concentrations.

Comparative mapping of novel simple sequence repeat markers in a hypertension-related region on rat chromosome 1.

~Department of Laboratory Medicine, Shimane Medical University, Izumo, 693, Japan 2Otsuka Department of International Preventive Nutritional Medicine, Kyoto University, Kyoto, 606, Japan 3Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, 606, Japan 4Institute of Laboratory Animals, Faculty of Medicine, Kyoto University, Kyoto, 606-01, Japan 5INSERM U. 358, Hopital St. Louis, 1, Av. Claude Vellefaux, Paris, 75475, France 6Wellcome Trust Centre for Human Genetics, University of Oxford, Windmill Road, Oxford, OX3 7BN, UK 7Department of Internal Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo, Japan 8Laboratory Animal Science and Toxicology Laboratories, Sankyo Co., Ltd., Fukuroi, Shizuoka, Japan

Increased Intracellular Ca2+ Is Not Coinherited With an Inferred Major Gene Locus for Hypertension (ht) in the Spontaneously Hypertensive Rat*

Hypertension is characterized by a complex mode of inheritance, consisting of the accumulation and interaction of major and minor genes. The existence of a single major gene locus (ht) has been demonstrated in the backcross analysis of spontaneously hypertensive rats (SHR) and normotensive Donryu rats. Intracellular Ca2+ concentration ([Ca2+]i) determines the tonus of vascular smooth muscle. It has been hypothesized that abnormal Ca2+ transport is an inheritable trait with profound influence on the development of hypertension. Backcross analysis between SHR and Donryu rats was performed to demonstrate ht and to dissect polygenic hypertensive traits through ht and abnormal intracellular Ca2+ metabolism. Among the parental strains, systolic blood pressure and thrombin-stimulated [Ca2+]i in platelets were significantly greater in SHR than in Donryu and F1 rats. The backcrossed rats were distributed into two clusters on a scattergram of blood pressure versus [Ca2+]i, demonstrating the existence of ht. The blood pressure level was correlated with thrombin-stimulated [Ca2+]i in each cluster. Increased [Ca2+]i in platelets was not coinherited with ht and was considered to be a minor inheritable hypertensive trait discriminated from ht. Therefore, [Ca2+]i in platelets is an inadequate marker for searching ht.

Genotypes of sarco(endo)plasmic reticulum Ca2+-dependent ATPase II gene in substrains of spontaneously hypertensive rats

Objective To search for a genetic marker of the sarco-(endo)plasmic reticulum Ca2+-dependent ATPase (SERCA) II gene in spontaneously hypertensive rats (SHRs) and to investigate differences in blood pressure and intracellular Ca2+ among some substrains of SHRs and Wistar–Kyoto (WKY) rats related to their SERCA II genotypes. Design and methods The coding region of the SERCA II gene was sequenced in SHRs. Blood pressure and intracellular Ca2+ concentration ([Ca2+]i) in platelets were measured in substrains of SHRs and WKY rats with different SERCA II genotypes. Results A point mutation that provided restriction fragment length polymorphisms (RFLPs) by Hindlll or Saul was found in the SERCA II gene. The polymerase chain reaction (PCR) products were digested by Hindlll in SHR substrains and WKY-Kyoto rats, whereas they were digested by Saul in normotensive strains and SHR-Toho. Among SHR-Kyoto, SHR-Toho, WKY-Kyoto and WKY-Charles River, the substrains with the Hindlll-digested SERCA II genotype showed slightly but significantly higher systolic blood pressure and augmented agonist-stimulated [Ca2+]i than those with the Saul-digested genotype. Conclusions RFLPs were found in the SERCA II gene. In the substrain analysis of SHRs and WKY rats, higher blood pressure and increased [Ca2+] were associated with the SERCA II genotype digested by W/ndlll. The SERCA II gene locus has the potential to contribute to the development of hypertension and abnormal intracellular Ca2+ metabolism in SHRs. These RFLPs in the SERCA II gene should be a useful genetic marker.

AUGMENTED Ca2+ MOBILIZATION IS A HYPERTENSIVE TRAIT DISCRIMINATED FROM A ‘MAJOR GENE’ IN BACKCROSS ANALYSIS BETWEEN SHR AND DONRYU RATS

1. Blood pressure and Ca2+ mobilization were significantly greater in SHR than in Donryu and F1 rats.