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Na,K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na+ transport during early postnatal development. The maximum Na(+)-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (ISCN) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats. ISCN was entirely inhibited by ouabain; the inhibitory constant was 0.1 mM in 10-day-old (young) and 0.4 mM in 90-day-old (adult) rats. The affinity of the Na,K pump for Na+ was higher in young (11 mM) than in adult animals (19 mM). The Na,K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (KI) was not changed during development (0.1-0.3 mM). Specific [3H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (KD) was 8 and 21 microM in young and adult rats, respectively. The Na+ turnover rate per single Na,K pump, which was calculated from ISCN and estimated density of binding sites per cm2 of tissue was 500 in adult and 6400 Na+/min.site in young rats. These data indicate that they very high Na+ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na+ of a single isoform of the Na,K pump. The development of Na+ extrusion across the basolateral membrane is not directly regulated by corticosteroids.

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.

Wet mass, resting membrane potential, frequency of miniature end-plate potentials and the concentration of [3H]ouabain-binding sites were studied after 7 days' immobilization of the rat soleus and extensor digitorum longus (EDL) muscles in the shortened or stretched position and after 3 and 7 days of remobilization. We observed that the loss of muscle mass by 37% in the rat soleus immobilized for 7 days in the shortened position is accompanied by a membrane depolarization of about 5 mV, a decrease in frequency of miniature end-plate potentials by 60 % and a decrease of [3H]ouabain binding by 25%. Only minor changes were found in stretched soleus and in shortened and stretched EDL. After 3 days of remobilization of stretched soleus the muscle mass, [3H]ouabain binding and miniature end-plate potential frequency recovered to control values but the resting membrane potential continued to decrease. All changes induced by immobilization disappeared on day 7 of remobilization.

The effect of 120-h sleep deprivation on the activity of selected enzymes of energy metabolism in skeletal muscle was studied in seven healthy volunteers. The results showed a significant decrease in the activity of malate dehydrogenase, citrate synthase, glycerol-3-phosphate dehydrogenase and lactate dehydrogenase. Triosephosphate dehydrogenase, hexokinase, and hydroxyacyl-CoA-dehydrogenase activities showed an insignificant decrease. The findings are indicative of (1) decreased aerobic oxidation capacity; (2) reduced function of reducing-equivalent carriers from cytosol across the mitochondrial membrane; (3) relative accentuation of the non-aerobic glycolytic pathway; (4) a prediabetic type of muscle metabolism.

The K+-induced hyperpolarization of Na-loaded mouse diaphragm muscle, enzymatic activity of Na,K-ATPase and 3H-ouabain binding to rat brain microsomes was measured in the presence of K+ channel blockers tetraethylammonium (TEA), tetrabutylammonium (TBA) and apamin. TBA, and to a lesser extent TEA in millimolar concentrations, inhibited the electrogenic effect of the Na,K pump, Na,K-ATPase activity, and 3H-ouabain binding. The inhibition of 3H-ouabain binding by TEA or TBA was more evident in the presence of ATP and Na+ ions. Apamin in nanomolar concentrations inhibited the electrogenic effect of Na,K pump and Na,K-ATPase but not the 3H-ouabain binding. The hyperpolarizing effects of insulin and NADH, but not that of noradrenaline, were also prevented by apamin. The inhibition of Na,K pump by TEA and TBA is apparently due to both competition with K+ for a binding site on the Na,K-ATPase and a reduction in the number of transporting sites. The site of action of apamin on Na,K-ATPase is different from that of tetra-alkylammonium compounds; it apparently decreases the turnover rate of the enzyme.

On crude membrane fractions of skeletal musccle, vanadyl (IV) and vanadate (V) compounds inhibited the membrane (Na + K + )-ATPase and neutral (K + -)p-nitrophenylphosphatase equally with K i 4×10 −8 mol.1 −1 . Only vanadate (V) inhibited significantly the muscle (Na + K + )ATPase with K i 1×10 −6 mol.1 −1 , whereas vanadyl (IV) ions were almost without effect. Extracellular application of both forms of vanadium failed to inhibit the electrogenic (Na + K + ) pump in intact mouse diaphragm fibres.

Differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene showed that vanadyl ions (VO2+) increased its limiting anisotropy (order parameter) in crude plasma membranes from brown adipose tissue of the golden hamster (Mesocricetus auratus). This was about 103 times larger than the effect of Ca2+ and was several times greater than the action of Co2+. Vanadate anions were without any effect. During the membrane treatment with VO2+, the rotational relaxational time of diphenylhexatriene did not change. This results suggest a possible positive influence of tetravalent vanadium on the stability of cell membranes.

Administration of cycloheximide in a single dose of 0.6 mg/kg to 7-day-old rats was used to induce short-term inhibition of protein synthesis at the period of brain 'growth spurt'. Measurement of the rate of [14C]lysine incorporation indicated that the initial inhibition of protein synthesis in the brain (by 75%) was released within about 12 h. The normal rate of protein synthesis was attained by 48 h after cycloheximide administration; there was no sign of protein synthesis stimulation. The estimation of [14C]thymidine incorporation into brain DNA showed that inhibition of DNA synthesis was greater and longer lasting in the forebrain and olfactory bulbs (by about 80%) than in the cerebellum (by about 40%). Similar differential inhibition of thymidine kinase activity was observed in the olfactory bulbs (by 75%) and cerebellum (by 30%) at 24 h after cycloheximide, suggesting that the formation of [14C]thymidine nucleotides may have been impaired. However, a retardation of DNA accumulation was found in the forebrain and cerebellum at 72 h after cycloheximide. Thus, the short-term inhibition of protein synthesis produced prolonged inhibition of DNA synthesis and altered cell proliferation in the developing brain.