Shigeyuki Takeda

Atrial Natriuretic Peptide Enhances IL-1β-Stimulated Nitric Oxide Production in Cultured Rat Vascular Smooth Muscle Cells

Objective: To elucidate the effect of atrial natriuretic peptide (ANP) on cytokine-induced nitric oxide (NO) production, we examined whether ANP affects IL-1β-stimulated NO production and inducible NO synthase (iNOS) mRNA expression in cultured rat vascular smooth muscle cells (VSMC). Methods: VSMC were incubated with test agents for 24 h. The nitrite concentration of the culture medium, a stable-end product of NO, was measured by the column reduction method. NOS mRNA expression was analyzed by Northern blotting. The intracellular cAMP content was measured by enzyme immunoassay. cAMP-dependent kinase (PKA) activity was determined by a PKA assay system. Results: IL-1β stimulated nitrite production in VSMC. ANP (10 nM to 1 mM) enhanced the nitrite production and iNOS mRNA expression in the presence of IL-1β. Both 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-br-cGMP) and dibutylyl adenosine 3′,5′-cyclic monophate enhanced IL-1β-induced nitrite production. The effects of these two nucleotide donors on the nitrite production were not additive, suggesting a common pathway. KT 5720, an inhibitor of PKA, abolished the enhancement of nitrite production by ANP and 8-br-cGMP, while KT 5823, an inhibitor of cGMP-dependent protein kinase, did not inhibit the enhancement. In the in vitro assay 8-br-cGMP increased PKA activity. Conclusions: ANP and cGMP enhanced IL-1β-induced NO production in VSMC. PKA rather than PKG may be involved in the enhancement.

Localization of Low-KM cAMP Phosphodiesterase in Rat Nephron Segments

To evaluate the roles of cAMP degradation on hormonal actions in the kidney, we examined the segmental distribution and activities of cAMP-phosphodiesterase (PDE), a key enzyme for cAMP hydrolysis, in dissected segments of the rat nephron and characterized the isozyme compositions with subtype-specific inhibitors. Summary of the nephron distribution of PDE activities showed that cAMP-PDE activities were detected in all nephron segments and the distal convoluted tubules (DCTs) had the highest activity. Both in proximal convoluted tubules (PCTs) and medullary collecting ducts (MCDs), more than 80% of the total cAMP-PDE activities were inhibited by a nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). In PCTs, rolipram (type-IV PDE inhibitor) was an equally potent inhibitor of IBMX, while 8-methoxymethyl-IBMX (MM-IBMX, type-I PDE inhibitor) and cilostamide (type-III PDE inhibitor) inhibited cAMP-PDE by 35 and 57%, respectively. In MCDs, inhibition by rolipram (40%) was less than that by MM-IBMX (70%) or cilostamide (66%). Rolipram potentiated the parathyroid hormone (PTH)-induced cAMP content in PCTs most effectively. Rolipram and MM-IBMX increased the vasopressin (AVP)-stimulated cAMP content equally in MCDs. Cilostamide had no effect on the cAMP content stimulated by PTH or AVP in PCTs and MCDs. These results indicate that PDE activities were unevenly distributed along the rat nephron and cAMP-PDE was composed of at least three isoforms, namely type I, III and IV in PCTs and MCDs. Among these, type-IV PDE was responsible for hydrolysis of cAMP in PCTs, and type-I and IV PDE were responsible for that in MCDs.

Application of Plasma Perfusion in Hepatic Failure

To evaluate the efficacy of plasma perfusion as a liver-assistance system, 17 patients with hepatic failure were treated with a total 37 sessions of plasma perfusion and 43 sessions of plasma exchange. For plasma perfusion, Plasma-flo AP-08-H was used as a plasma separator, and the separated plasma was passed through an adsorber, Bespore UPC-III (Jap.Med.Suppl., Hiroshima) or BR 350 (Asahi Med. Co., Tokyo). The mean treated plasma volume was 5.0 liters. The mean proportion of bilirubin removed by routine plasma exchange was 29% with a mean of plasma exchange volume of 2.1 L. In the plasma perfusion with Bespore and BR, the removal rates were 37% and 42%, respectively. The plasma ammonia level decreased by 16% (from 121 to 97 mumol/L) with plasma exchange. Likewise, plasma perfusion decreased the plasma ammonia level from 75 to 56 mumol/L with an average decrement rate of 23%. The capacity for removal of amino acids was analyzed in terms of the change in the Fisher score ([Val+Leu+Ileu]/[Tyr+Phe]). The Fisher score was improved by plasma perfusion from 1.8 to 2.4. Thus, we concluded that in terms of removal of bilirubin, ammonia and amino acids, plasma perfusion was as efficient a therapy as plasma exchange. However, further clinical evaluation will be required before this procedure can be applied for the treatment of hepatic failure.

Endothelin‐1 affects AVP‐stimulated cAMP but not ANP‐induced cGMP productions in cultured rat IMCD cells

Summary: The present study was undertaken to explore a potential interaction of endothelin‐1 (ET‐1) on vasopressin (AVP)‐dependent cyclic 3′,5′‐adenosine monophosphate (cAMP) and atrial natriuretic peptide (ANP)‐dependent cyclic 3′,5′‐guanosine monophosphate (cGMP) metabolisms in primary cultured rat inner medullary collecting duct (IMCD) cells. Endothelin‐1 did not affect ANP‐stimulated cGMP accumulation in the presence or absence of 3‐isobutyl‐1‐methylxanthine (IBMX). Levels of 10−10 to 10−7 mol/LET‐1 showed a dose‐dependent inhibition of the AVP‐dependent cAMP accumulation in the presence of IBMX and 10−7 mol/LET‐1 inhibited the cAMP generation by 34 ± 5.3% (n= 5, n= 20, P<0.01). Endothelin‐1 did not affect the cAMP generation either by 100 ng/mL cholera toxin or by 10−4 mol/L forskolin. Endothelin‐1 failed to inhibit the cAMP generation in the presence of 100 ng/mL pertussis toxin (PTX). the inhibitory effect of ET‐1 was reversed by 10−8 mol/L staurosporin (SSP), a protein kinase C (PKC) inhibitor. Furthermore, this inhibitory effect of ET‐1 was mimicked by 10−8 mol/L phorbol 12‐myristate 13‐acetate (PMA), an activator of PKC. A dose of 5 × 10−6 mol/L indomethacin did not affect this inhibitory effect of ET‐1. From these results, we suggest that the effect of ET‐1 on cyclic nucleotides metabolism seem to be selective. Endothelin‐1 did not affect the cGMP generation by ANP, whereas it inhibited cAMP production by AVP via PTX and SSP sensitive pathway in cultured rat IMCD cells.