Natriuretic peptides (NPs), mainly produced in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and kidney (urodilatin), decrease blood pressure and increase salt excretion. These functions are mediated by natriuretic peptide receptors A and B (NPRA and NPRB) having cytoplasmic guanylyl cyclase domains that are stimulated when the receptors bind ligand. A more abundantly expressed receptor (NPRC or C-type) has a short cytoplasmic domain without guanylyl cyclase activity. NPRC is thought to act as a clearance receptor, although it may have additional functions. To test how NPRC affects the cardiovascular and renal systems, we inactivated its gene (Npr3) in mice by homologous recombination. The half life of [ 125 I]ANP in the circulation of homozygotes lacking NPRC is two-thirds longer than in the wild type, although plasma levels of ANP and BNP in heterozygotes and homozygotes are close to the wild type. Heterozygotes and homozygotes have a progressively reduced ability to concentrate urine, exhibit mild diuresis, and tend to be blood volume depleted. Blood pressure in the homozygotes is 8 mmHg (1 mmHg ‫؍‬ 133 Pa) below normal. These results are consistent with the sole cardiovascular͞renal function of NPRC being to clear natriuretic peptides, thereby modulating local effects of the natriuretic peptide system. Unexpectedly, Npr3 ؊͞؊ homozygotes have skeletal deformities associated with a considerable increase in bone turnover. The phenotype is consistent with the bone function of NPRC being to clear locally synthesized CNP and modulate its effects. We conclude that NPRC modulates the availability of the natriuretic peptides at their target organs, thereby allowing the activity of the natriuretic peptide system to be tailored to specific local needs.

Klotho, a regulatory factor implicated in countering the aging process, has been reported to ameliorate endothelial dysfunction in vivo. To clarify whether Klotho protein directly affects endothelial cell function, we studied the effects of membrane-form Klotho on manganese superoxide dismutase (Mn-SOD) expression and nitric oxide production in human umbilical vein endothelial cells (HUVEC). We incubated HUVEC with conditioned medium from COS-1 cells transfected with expression vector, pCAGGS-klotho (Klotho-CM) or a recombinant, purified 6His-tagged Klotho protein. Both Klotho-CM and 6His-tagged Klotho protein enhanced Mn-SOD expression by approximately two-fold, partially via activation of the cAMP signaling pathway. Furthermore, Klotho-CM increased nitric oxide production, which also contributed to the up-regulation of Mn-SOD. Using the oxidation-sensitive dye dihydroethidium, we found that Klotho inhibited angiotensin II-induced reactive oxygen species production in HUVEC. These findings provide new insights into the mechanisms of Klotho action and support the therapeutic potential of membrane-form Klotho to regulate endothelial function.

Either low or high levels of insulin suppressed angiotensinogen and renin expression, however, high doses of insulin stimulated ACE activity in cultured human aortic EC. This may indicate that insulin regulates vascular cell growth and endothelial function via bifunctional modification of the vascular angiotensin generation.

A cDNA clone encoding cytochrome P‐45011β of rat adrenal has been cloned and sequenced using a bovine P‐45011β cDNA insert (pcP‐450(11β)‐2; (1987) J. Biochem. 102, 559–568) as a probe. The nucleotide sequence contains an open reading frame sufficient to encode the entire amino acid sequence of a P‐45011β precursor protein consisting of 499 amino acids including an extension peptide of 24 amino acids at the NH2‐terminus. The cDNA contains 1247 nucleotides at the 3′‐noncoding region including 51 nucleotides of poly A, but lacks the 5′‐noncoding region. The deduced amino acid sequence shows 61% similarity to that of bovine P‐45011β. Putative binding sites for heme and steroid are highly conserved among steroidogenic P‐450s of known structure.

Homozygous Klotho mutant (kl-/-) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl-/- mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D3 1alpha-hydroxylase in the kidney. We studied 25-hydroxy-vitamin D3 1alpha-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D3 1alpha-hydrox-ylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D3 1alpha-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.