Shuzo Kudo

Effects of local anesthetics on calmodulin-dependent guanylate cyclase in the plasma membrane of tetrahymena pyriformis

A highly purified preparation of Tetrahymena calmodulin activated a membrane-bound guanylate cyclase by more than 40-fold. This activation of guanylate cyclase by calmodulin was inhibited completely by local anesthetics such as dibucaine, tetracaine, lidocaine and procaine at concentrations that had no appreciable effect on the activities of basal guanylate cyclase (without calmodulin) and adenylate cyclase. The inhibition by dibucaine of calmodulin-mediated activation of the enzyme activity was not reversed by calcium but was partially overcome by increasing the concentration of calmodulin. Kinetic analysis of local anesthetic-induced inhibition of activation of guanylate cyclase demonstrated a mixed type of antagonism. These results suggest the possibility that the inhibition of calmodulin-dependent guanylate cyclase resulted, in part, from interaction of the drugs with calmodulin.

Specificity of Tetrahymena calmodulin in activation of calmodulin-regulated enzymes

Calmodulin was discovered in rat brain [ 1,2] and subsequently identified in a variety of tissues. It now appears to play a role of multifunctional intracellular Ca2+ receptor [3-51. It is thought that in general the structure and function of c~modnlin is conservatively maintained throughout the animal and plant kingdoms during evolution. We have described the isolation of calm~ulin from unicellular eukaryote, Tetrahymena pyriformis and found that it could fully activate guanylate cyclase in this organism in a Ca’+-dependent fashion [6-lo]. However, the activation of guanylate cyclase was specifically attributable to the calmodulins from Te~ra~y~ena and Paramecium [9,10]. Moreover, comparison of the primary structure of calmodulin from bovine brain [ 1 lf and Tetra~yme~a 1121 shows 12 amino acid differences between the 2 proteins. Therefore, calmodulin may vary in structure and function. Here, we have compared the functional properties between the two calmodulins from Tetrahymena and bovine brain. The data demonstrate that both Tetra~ymena and bovine brain calmodulins are similar in their potency to activate various calmodulin-dependent enzymes such as rat brain adenylate cyclase, myosin light-chain kinase, erythrocyte (Ca’+ + Mg2+)-ATPase and plant NAD kinase. Calmodulins were prepared from Tetrahymena and bovine brain as in [8]. A th~rmotolerant strain NT-l of Tetrahymena pyriformis was grown at 395°C in an enriched proteose peptone medium as in [ 131. The plasma membrane fraction which contains guanylate cyclase was prepared as in [13]. Calmodulin-dependent phosphodiesterase was prepared from bovine brain as in [14]. Adenylate cyclase was solubilized from washed particulate preparation of rat cerebrum using 1% Lubrol PX and elute from Ultro Gel AcA 34 column with 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 olo Lubrol PX, 0.1 mM EGTA, 0.1 M sucrose and 1 mM EDTA as in [15]. Myosin light-chain kinase was prepared from chicken gizzard as in [ 161. The light-chain kinase from chicken gizzard myosin was prepared essentially as in [ 171. The light chain was separated from calmodulin by DEAF&cellulose column chromatography [18], Erythrocyte membranes for (Ca2+ + Mg2+)-ATPase assay were prepared as in [ 191. Membranes were freeze-thawed 3 times just prior to incubation for ATPase assay. The preparation of NAD kinase was obtained from pea seedling (Pisum sativum) grown for 12 days in natural light at 20-25’C. The NAD kinase was prepared as in [ZO]. Guanylate cyclase activity was assayed as in [9], cyclic AMP phosphodiesterase activity as in [8]

Growth-associated changes in cyclic nucleotide enzymes in Tetrahymena: Involvement of calmodulin

The activities of enzymes responsible for cyclic nucleotide synthesis and degradation were studied during growth of Tetrahymena pyriformis, strain GL. The significant changes in the enzyme activities involving the cyclic GMP system were observed. Moreover, a close relationship was found to exist between the guanylate cyclase activity and the calmodulin level. While the guanylate cyclase activity was greatly diminished by trifluoperazine, a potent inhibitor of calmodulin, the activities of other enzymes were unaffected. These results suggest that the guanylate cyclase activity may be regulated by calmodulin level during growth in T. pyriformis.

Growth-dependent changes of guanylate and adenylate cyclase activities in cilia and cell bodies of Tetrahymena pyriformis

The calmodulin-dependent guanylate cyclase of Tetrahymena pyriformis was shown previously to be localized in surface membranes (ciliary and pellicular membranes) (Kudo, S, Nakazawa, K, Nagao, S & Nozawa, Y, Japan j exp med 52 (1952) 193) [21], whereas in a recent report Schultz et al, (Schultz, J E, Schonefeld, U & Klumpp, S, Eur j biochem 137 (1983) 89) [12] demonstrated the localization of this enzyme in ciliary membrane, arguing against its presence in pellicular membrane. To examine the discrepancy, the activities of guanylate and adenylate cyclases were examined in cilia and cell bodies of Tetrahymena pyriformis during transition from early log to stationary growth phase. The guanylate cyclase activity in the cell bodies increased significantly with growth of age, while in cilia the activity was rather consistent. In contrast, adenylate cyclase did not show any growth-dependent activity changes in both cilia and cell bodies. The increase of guanylate cyclase activity was not related to the increase of its activator calmodulin, because the change in enzyme activity could not be negated by addition of a saturating amount of calmodulin. These results suggest that the content of guanylate cyclase itself would be increased in the cell bodies during growth.

Interaction of calcium-binding proteins with calmodulin-dependent guanylate cyclase in Tetrahymena plasma membrane.

Addition of bovine brain calmodulin and S-100 inhibited Tetrahymena calmodulin-induced stimulation of guanylate cyclase, but they did not affect enzymatic activity in the presence of calcium alone. Troponin C shows little effect on the cyclase activity regardless of the presence or absence of Tetrahymena calmodulin. The inhibitory effects of brain calmodulin and S-100 were overcome by the addition of Tetrahymena calmodulin, but not by calcium. Both calmodulins from Tetrahymena and bovine brain elicited stimulation of heart phosphodiesterase, while troponin C and S-100 did not affect the phosphodiesterase activity in the presence and absence of Tetrahymena calmodulin.

Inhibitory effects of calmodulin antagonists on plasma membrane cyclases in tetrahymena: Calmodulin-dependent guanylate cyclase and calmodulin-independent adenylate cyclase

Trifluoperazine was shown previously to inhibit the activation of Tetrahymena guanylate cyclase activity by calmodulin [S. Nagao, S. Kudo and Y Nozawa, Biochem. Pharmac. 19, 2709 (1981)]. The present paper reports that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), another representative calmodulin inhibitor, inhibited the calmodulin-induced activation of the guanylate cyclase, and that trifluoperazine and W-7 also inhibited Tetrahymena adenylate cyclase. The adenylate cyclase activity was found to be present in a membrane-bound form and not to be influenced by calmodulin. The inhibitions of the adenylate cyclase activity by these agents were dose-dependent and not Ca2+-dependent. These findings suggest that the inhibitory actions of these drugs may not necessarily be specific for calmodulin-dependent enzymes in T. pyriformis.

Characterization of cyclic AMP and cyclic GMP phosphodiesterases in Tetrahymena cilia

Abstract 1. 1. The activities of cyclic AMP and cyclic GMP phosphodiesterases were found to be associated with both soluble and particulate fractions in Tetrahymena cilia. 2. 2. The properties of these enzymes in ciliary fractions were similar to those of cell bodies in K m , pH dependency, inhibition by methylxanthine and lack of a requirement for Mg2+. 3. 3. These enzyme activities of cell bodies of behavioral mutants were higher than those of wild type, while the enzyme activities of cilia did not show differences in both wild and mutant cells.