Effects of guanine nucleotides and parathyroid hormone on inositol 1,4,5‐trisphosphate metabolism in canine renal cortical tubular cell membranes

Parathyroid hormone (PTH) and guanosine 5′‐ O ‐(3‐thiotriphosphate) (GTP‐γS) increase levels of the second messenger inositol 1,4,5‐trisphosphate (IP 3 ) and other inositol phosphates (IP) in several membrane preparations of PTH‐responsive cells. We present evidence here indicating that in a membrane preparation of canine renal cortical tubular cells bPTH‐(1–84), bPTH‐(1–34), [N‐Leu 8,18 Tyr 34 ]bPTH‐(3–34)NH 2 , and the human PTH related peptide fragment hPTHrP‐(1–34)NH 2 all increase levels of inositol phosphate (IP) but [Tyr 34 ]‐bPTH‐(7–34)NH 2 and hPTHrP‐(7–34)NH 2 have no significant effects on IP accumulation. Increases in IPs are generally attributed to increased formation of IPs and appear to be mediated by a G protein. However, increased levels of IPs may also result from inhibition of the phosphatases that are responsible for their metabolism. We investigated the effect of PTH and GTP‐ γ S on the metabolism of IP 3 in canine renal cortical tubular membranes. These membranes rapidly metabolize [ 3 H]IP 3 (47% at 15 s). Decreases in [ 3 H]IP 3 at all time points are accounted for quantitatively by increases in the sum of its breakdown products: [ 3 H]IP 2 , [ 3 H]IP 1 , and [ 3 H]inositol. After 5 minutes of exposure to membranes, the vast majority of [ 3 H]IP 3 (84%) is converted to its terminal metabolite, [ 3 H]inositol. GTP‐ γ S (100 μM) inhibits the amount of [ 3 H]IP 3 metabolized in 15 s by 70% and reduces the amount of [ 3 H]inositol ultimately formed in 5 minutes by 64%. ATP‐γS, ATP, and 2,3‐bisphosphoglycerate (100 μM) also inhibit [ 3 H]IP 3 hydrolysis in this preparation. The results demonstrating that nucleotides, such as GTP‐ γ S, at concentrations used to demonstrate G protein‐mediated increases in IP 3 formation, can inhibit the metabolism of IP 3 suggest that their effect to increase levels of IP 3 may be due in part to inhibition of IP 3 metabolism. In contrast, PTH‐(1–34), PTH‐(3–34), and hPTHrP‐(1–34) (1 μM) do not reduce the amount of [ 3 H]IP 3 metabolized in 15 s, indicating that the site of action of PTH on IP 3 levels is predominantly at steps before the hydrolysis of IP 3 .