Tag Archives: colonic smooth muscle

Assessment of the effect of monohydroxy alcohols, unsaturated fatty acids, organophosphate compounds on the enzymatic ATP-hydrolysis in the cell membranes of the smooth muscle of the rat colon

A. A. Kaplia, S. V. Midyk1, S. V. Khyzhnyak1

1National University of Life and Environmental Sciences of Ukraine, Kyiv;
e-mail: kaplyaalex@gmail.com

The differences of the structural-functional stability of ATP-hydrolases to the action of the short-chain aliphatic monohydroxy alcohols and long-chain unsaturated fatty acids (UFAs) with wide range of the membrane-acting effectiveness were evaluated in the colonic smooth muscle (CSM) cellular membranes. It was shown that Mg2+,ATP-hydrolase was more resistant to all tested aliphatic compounds than Na+,K+-ATPase. There was  a clear relationship between  inhibitory efficiency, chain length (number of carbons) increase and relative polarity decrease  in the series of straight-chain alcohols. Secondary alcohol 2-propanol turned out to be the weaker inhibitor than primary alcohol 1-propanol. The I50 value (мМ) for ATРases decreased in the following succession: ethanol > 2-propanol > 1-propanol > 1-butanol. UFAs were the stronger inhibitors (I50 of μM range) than alcohols (mM range). Oleic acid inhibited Na+,K+-ATPase with lower efficiency than was detected for arachidonic acid according to the differences in double bonds number; I50 for Mg2+,ATP-hydrolase activity was the same for studied UFAs. As well, the organophosphate pesticide fenamiphos proved to be a weak ATPase inhibitor. Comparison of the enzymatic pair of the CSM ATP-hydrolases seems applicable for evaluation of the specific membrane-acting effect of bioactive compounds on Na+,K+-ATРase activity from the standpoint of its role in mediating of electro-mechanical coupling in intestinal smooth muscles.

Different sensitivity of Na(+),K(+)-ATPase and Mg(2+)-АТРase to ethanol and arachidonic acid in rat colon smooth muscle under pretreatment of cellular membranes with Ds-Na

A. A. Kaplia

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kaplya@biochem.kiev.ua

The methodological procedure provides the detection of the relatively high Na+,K+-ATPase functional activity in the crude cellular membranes of rat colon smooth muscle (CSM) following standard detergent pretreatment (with Ds-Na vs digitonin). It includes the essential discrete steps: detergent membrane permeabilization under optimal detergent/protein ratio and active site protection by ATP (for Ds-Na) prior enzymatic reaction with substantial detergent dilution far below critical micelle concentration in the ATPase medium. The high level of the Na+,K+-ATPase activity, originally detected in CSM, did not differ for two detergents and was comparable with ouabain-resistant Mg2+,АТР-hydrolase activity, The features of ATPase protein-lipid complexes were evaluated by the enzyme sensitivity to the effect of ethanol and arachidonic acid with different membrane disordering effectiveness. The long-chain fatty acid is a more effective inhibitor as compared with aliphatic alcohol for both ATPases. Mg2+,АТР-hydrolase appeared to be much more resistant to inactivation than Na+,K+-ATPase. The data reflect the possible differences in lipid dependence of two enzymatic systems due to the peculiarities of the structural arrangement in membrane and importance of the hydrophobic microenvironment for mechanism of catalysis. Thus, the data represent the approach to the simple and reliable Na+,K+-АТРase activity determination in nonpurified CSM membranes, acceptable for different tissues and appropriate for quantitative comparison in pathophysiological studies and for testing the impact of diverse  effectors on Na+,K+-АТРase.

The influence of heavy metal ions, spermine and sodium nitroprusside on ATP-hydrolases of cell membranes of rat colon smooth muscle

A. A. Kaplia

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kaplya@biochem.kiev.ua

The specific features of functional lability of the rat colon smooth muscle (CSM) АТР-hydrolases were studied. Na+,K+-AТРase activity is effectively inhibited by divalent ions of both transition (≥ 0,1 µM) and nontransition (≥ 1 µM) heavy metals in succession by efficiency: Cu2+ > Fe2+ ≥ Cd2+ (10 µM). Polyamine spermine (0,5-1,0 mM) is a weak  Na+,K+-AТРase inhibitor at saturation concentrations of ions and substrate. Sodium nitroprusside (1 mM) as nitric oxide-generating compound exhibits weak Na+,K+-AТРase inhibition only after prolonged preincubation with membranes. Mg2+-АТР-hydrolase activity in all cases is much more resistant to studied agents. Considering the example of the CSM Na+,K+-AТРase it is assumed that  enzyme has specific biochemical features that contribute to its role as a potential target and redox-sensor, mediating the pathological mechanisms of heavy metal intoxication and cell oxidative damage.

The influence of iron ions on ATP-hydrolases activity of cell membranes of rat colon smooth muscle and kidney

A. A. Kaplia

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kaplya@biochem.kiev.ua

To elucidate the specific features of the АТР- hydrolases structural resistance in the membrane under the action of the prooxidants: Fe2+ and hydrogen peroxide, and N-ethylmaleimide (NEM)  the colonic smooth muscle (CSM) Na+,K+-AТРase activity was compared with activities of the corresponding Mg2+-АТР-hydrolase and ATP-ases from kidney medullar layer of rats. The inhibition study of the CSM Na+,K+-AТРase by divalent iron shows the decrease of the activity by 30% at 0.1 µM FeSO4 and in the range of 0.1-10 µM – to 45% of residual activity. When comparing with kidney enzyme (represents exclusively α1-isozyme) the CSM Na+,K+-AТРase sensitivity to Fe2+ is reliably higher at its submicromolar concentration. CSM Mg2+-АТРase is much more resistant to iron ions effect, than kidney one. However for two tissues Mg2+-АТРase activi­ty is always more resistant as compared with corresponding Na+,K+-AТРase activity. Against 1 mM EGTA Na+,K+-AТРase and Mg2+-АТРase activities of GMOK and kidneys are equally insensitive to effect of hydrogen peroxide in concentration up to 1 mM. But in the presence of 20 µM FeSO4 in the concentration range of 1 nМ – 1 mM of Н2О2 the Na+,K+-AТРase is inhibited to greater extent, than Mg2+-АТРase activity. NEM sensitivity of the two АТР-hydrolase systems corresponds to prooxidant sensitivity that indicates the distinct importance of SH-groups for their functioning. It is concluded that Na+,K+-AТРase can serve as a marker of membrane sensitivity to oxidation, Mg2+-АТРase is resistant to oxidation and can be considered as criterion of the oxidation resistance when comparing  membrane enzyme complexes, especially in GMOK.