Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.

Inhibition of T-lymphocyte activation by amiloride.

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.

Difference in calcium sensitivities of the sodium transporting systems in the nervous system of susceptible and kdr-resistant houseflies, Musca domestica L.

1. The synaptosomal preparations from the kdr-resistant strains have lower responsiveness to externally applied Ca2+ than the susceptible counterparts with respect to their Na+/Ca2+ exchange, Na+ uptake, Na+-Ca2+ protein kinase-phosphatase and its phosphorylation activities. 2. In vivo toxicity tests support the above in vitro observation in that the kdr-resistant strain shows a significant cross-resistance to agents known to affect the Na+ channel operations, those which increase intracellular Ca2+ or act as effective Ca2+ substitutes. 3. Such evidence indicates a possibility of a causal relationship between kdr resistance and the reduction of Ca2+ sensitivity by these ATP-utilizing systems including the Na+ transporting systems in the resistant insects.

Interaction of DDT and pyrethroids with calmodulin and its significance in the expression of enzyme activities of phosphodiesterase.

To understand the significance of the inhibitory action of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and pyrethroid insecticides on calmodulin, a universal Ca2+ binding protein, a bovine heart phosphodiesterase-calmodulin system was studied. It was found that, at concentrations of less than 10(-5) M, the inhibitory action of DDT of the phosphodiesterase was due entirely to its action on calmodulin alone. Cypermethrin was less potent than DDT, but it also affected only calmodulin. Permethrin was the most potent inhibitor affecting calmodulin and, to a lesser extent, phosphodiesterase. The inhibitory action of these insecticides on calmodulin raises a possibility that many unsuspected Ca2+-related systems are being affected by these insecticidal chemicals, as calmodulin is known to play vital roles in many biological reactions dependent upon Ca2+. These include modulation of phosphodiesterase, neurotransmitter release, adenylate cyclase, Ca2+-dependent protein kinase, myosine light chain kinase and various membrane phosphorylation systems.

Reduced calcium sensitivity of the sodium channel and the Na+/Ca2+ exchange system in the Kdr-type, DDT and pyrethroid resistant German cockroach, Blattella germanica.

To understand the biochemical cause of nerve insensitivity to DDT and pyrethroids two cation regulating systems, the Na+/Ca2+ exchange and the Na+ channel in the membrane preparation from the central nervous system of susceptible and resistant strains of the German cockroach were examined. Both systems from resistant insects have lower affinities to Ca2+ than those from their susceptible counterparts. The resistant strains show a prominent cross-resistance to all agents affecting calcium regulation (particularly A23187) and Na+ channel operations in vivo, indicating that the basic cause of the reduced nerve insensitivity in resistant insects is related to the reduction in calcium sensitivity in these systems.

Effect of chlordecone on pH and temperature dependent substrate activation kinetics of rat brain synaptosomal ATPases.

Chlordecone, a polycyclic chlorinated insecticide known as Kepone, inhibited the activities of (Na+-K+)ATPase and Mg2+-ATPase in rat brain synaptosomes. Altered pH and specific activity curves for both enzymes demonstrated significant inhibition by chlordecone in buffered acidic, neutral and alkaline pH ranges. Noncompetitive inhibition with respect to activation by ATP in the case of (Na+-K+)ATPase was indicated by altered Vmax values with no significant change in Km values at any pH studied, except at pH 9.5. Mg2+-ATPase was inhibited uncompetitively as evidenced by altered Vmax and Km values. The activities of both ATPase were decreased in the presence of chlordecone at higher temperatures. Activation energy (delta E) values were found to be decreased significantly in the presence of chlordecone at 37 degrees. Arrhenius plots of both ATPases preincubated with chlordecone were found to be nonlinear. In the presence of chlordecone, Vmax was decreased without significant change in Km values for (Na+-K+)ATPase at all temperatures, suggesting a noncompetitive type of inhibition. In the case of Mg2+-ATPase, similar noncompetitive type inhibition was obtained at 27 degrees but not at 32 and 37 degrees. The kinetic data in general suggest that the chlordecone inhibited (Na+-K+)ATPase noncompetitively and Mg2+-ATPase uncompetitively at all pHs and temperatures studied. The present data suggest that inhibition of (Na+-K+)ATPase and Mg2+-ATPase, the two membrane-bound enzymes in synaptosomes, by chlordecone is temperature dependent and pH independent.

Effect of chronic herbicide intoxication on in vivo activities of certain enzymes in the liver of freshwater fish Nemachelius denisonii (day).

Effect of 120 days of continuous exposure of three sublethal concentrations (1/2, 1/4 and 1/8 fractions of 96 h LC50) of Basalin on alkaline phosphatase (Alk P), acid phosphatase (Acid P), lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) activities in the liver of Nemacheilus denisonii were studied. Alk P, Acid P, LDH, and GPT activities were significantly inhibited, but GOT activities were not significantly altered. More inhibition was observed with the higher concentration, but Acid P, LDH, and GPT activities were significantly inhibited in all three sublethal concentrations.