Interaction of smooth muscle relaxant drugs with calmodulin and cyclic nucleotide phosphodiesterase

Some smooth muscle relaxant drugs with an unknown mechanism of action have been tested for their interaction with calmodulin and with calmodulin-induced cyclic nucleotide phosphodiesterase (PDE) activity. The affinity of these drugs for calmodulin does not parallel their inhibitory effect on the calmodulin activation of PDE. The lack of parallelism could be due to a binding of the drugs to different sites on calmodulin; furthermore a binding of papaverine, octylonium bromide and felodipine to PDE molecule might also be considered to explain their inhibitory effect on PDE basal activity. The myolytic effect of octylonium bromide and pinaverium bromide may be due to their interaction with calmodulin-dependent systems.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Calmodulin mediates melatonin cytoskeletal effects

In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10^–9 M) cytoskeletal effects are mediated by its antagonism to Ca²⁺-calmodulin. At higher concentrations (10^–5 M), non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca²⁺-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

Summary The effect of calcium on Na, K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10^–6mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,-K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.The expert technical assistance of Mrs Paula Jarvie is gratefully acknowledged. Thanks are due also to Professor Philip Kuchel for assistance with the calculations to determine the concentrations of metal-ligand complexes in the experimental media.     

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors

The mechanism of the in vitro inhibition of Ca2+-, phosphatidylserine-dependent protein kinase C (PK-C)2 by the purified holo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that the PK-C-inhibitory action of holo-cRBP and holo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; the reduced phosphorylation of the holo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and the structure-function activity of the retinoids and the specific interaction of these compounds with their binding proteins are important in blocking the activity of PK-C. These observations suggest that the inhibitory effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.     

Failure of calcium to stimulate Na,K-ATPase in the presence of EDTA

The effect of calcium on Na,K-ATPase activity of rat brain homogenates and its modification by the chelating agent EDTA has been investigated. In the absence of EDTA, free calcium (approximately 10(-6) mol/l) stimulates Na,K-ATPase activity; in the presence of EDTA the same concentration of free calcium is without effect on the enzyme. In the absence of EDTA the stimulation by calcium of Na,K-ATPase activity is enhanced by the additional presence of calmodulin but in the presence of EDTA, even when calmodulin is added to excess, calcium still fails to stimulate the enzyme. The possibility that EDTA interferes with an interaction between a calcium-calmodulin complex and Na,K-ATPase is discussed.     

Effects of [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) on cyclic 3',5'-nucleotide formation and phosphodiesterase activity.

The orally-effective antiallergic compound [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) exhibited a potency equivalent to or 3 times less than theophylline in inhibiting guinea-pig lung and beef heart PDE, respectively, AY-25,674 did not affect the basal activity of guinea-pig lung adenyl cyclase. Although part of the antiallergic activity of AY-25,674 may be due to the ability to elevate cyclic AMP levels by PDE inhibition, other modes of action appear to be of greater relevance.     

Ca2+/Calmodulin-dependent Protein Kinases

In this article the calcium/calmodulin-dependent protein kinases are reviewed. The primary focus is on the structure and function of this diverse family of enzymes, and the elegant regulation of their activity. Structures are compared in order to highlight the conserved architecture of their catalytic domains with respect to each other as well as protein kinase A, a prototype for kinase structure. In addition to reviewing structure and function in these enzymes, the variety of biological processes for which they play a mediating role are also examined. Finally, how the enzymes become activated in the intracellular setting is considered by exploring the reciprocal interactions that exist between calcium binding to calmodulin when interacting with the CaM-kinases.     

Effects of [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) on cyclic 3′,5′-nucleotide formation and phosphodiesterase activity

Summary The orally-effective antiallergic compound [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) exhibited a potency equivalent to or 3 times less than theophylline in inhibiting guinea-pig lung and beef heart PDE, respectively. AY-25,674 did not affect the basal activity of guinea-pig lung adenyl cyclase. Although part of the antiallergic activity of AY-25,674 may be due to the ability to elevate cyclic AMP levels by PDE inhibition, other modes of action appear to be of greater relevance.The authors acknowledge the excellent technical assistance of Ms M.T. Silvestre.     

Calcium transport by red blood cell membranes from young and adult cattle

Summary It is shown using inside-out membrane vesicles that cattle red cells extrude calcium by means of a calmodulin sensitive Ca-pump whose activity is high in calves and extremely low in adult cows. The deline is not due to loss of calmodulin susceptibility nor to a drop in Ca-affinity.     

Possible involvement of calmodulin in apical constriction of neuroepithelial cells and elevation of neural folds in the chick

Chlorpromazine and trifluoperazine HCl, antipsychotic drugs known to bind to calmodulin, reversibly inhibited elevation of neural folds by interfering with the contractile activity of apical microfilament bundles in developing chick neuroepithelial cells.     

Calcium transport by red blood cell membranes from young and adult cattle

It is shown using inside-out membrane vesicles that cattle red cells extrude calcium by means of a calmodulin sensitive Ca-pump whose activity is high in calves and extremely low in adult cows. The decline is not due to loss of calmodulin susceptibility nor to a drop in Ca-affinity.     

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Lack of effect of dihydroergotamine on endothelial and smooth muscle cell proliferation and endothelial cell prostanoid production

Summary The most important effect of dihydroergotamine is venoconstriction, but certain metabolic effects and changes in vessel prostanoid activity have also been suggested. In this study endothelial cell production of 6-keto PGF₁ and TxB₂ was quantitated in vitro. No evidence of altered prostanoid production was noted after incubation with dihydroergotamine (exposure ranging from 5×10^–3 to 5×10^–7 g/l). Similarly, no effect of dihydroergotamine on the growth rates of endothelial cells or smooth muscle cells in vitro was documented.     

Ergot alkaloids and phosphodiesterase; 'in vitro' activities in several rat brain areas

The 'in vitro' activity of ergotamine, dihydroergocristine, dihydroergocornine and dihydroergocryptine on the phosphodiesterase system at low and high Km in several rat brain areas was examined. These drugs were found to exert an inhibitory effect in all the areas examined with regard to both systems, and particularly on low substrate concentration phosphodiesterases.     

Possible involvement of calmodulin in apical constriction of neuroepithelial cells and elevation of neural folds in the chick

Summary Chlorpromazine and trifluoperazine HCl, antipsychotic drugs known to bind to calmodulin, reversibly inhibited elevation of neural folds by interfering with the contractile activity of apical microfilament bundles in developing chick neuroepithelial cells.We wish to thank C. Hess for her technical assistance. This study was supported in part by grants from the Busch Fund (Rutgers University), UMDNJ Foundation and Kapnek Fund.     

Influence of the carbohydrate moiety on the growth inhibitory activity and adhesiveness of 3T3 cell plasma membranes

Summary Treatment of 3T3 cell plasma membranes with glycosidase enzymes decreased their ability to inhibit cell growth and also decreased their binding to 3T3 cells. This suggests that carbohydrate is required for complete function of inhibitory activity and that inhibition is associated with membrane adhesion.