A simple yet precise microassay for as little as 5 ppmoles of 3 ',5 '-cyclic adenosine monophosphate or 3 ',5 '-cyclic guanosine monophosphate has been developed through a modification of the isotope dilution principle. The hydrolysis of tritium-labeled cyclic nucleotides by a uniform phosphodiesterase reaction is diminished by the addition of nonlabeled cyclic nucleotides. Cyclic nucleotides are isolated from tissue samples by perchloric acid extraction, absorption and elution from small anion-exchange columns, and lyophilization. A simple preparation from rat brain contains both 3 ',5 '-cyclic adenosine monophosphate and 3 ',5 '-cyclic guanosine monophosphate phosphodiesterase activities. Enzymz reaction mixtures include snake venom to provide an excess of 5 '-nucleotidase activity. The nucleosides produced in the coupled enzyme system are separated from unreacted substrate by treatment
A B S T R A C TAnalyses of ashed muscle tissue show that the uptake of Ca 46 by isolated frog heart ventricles from normal Ringer's solution containing 1 mM Ca reaches a maximum value in about 30 minutes of perfusion which is not exceeded after 3 hours of perfusion. The average amount of this labeled Ca taken up from normal Ringer's is 0.7 mM/kg, wet weight of muscle. In contrast to this, the amount of labeled Ca taken up by ventricles perfused with Kfree Ringer's increases at a linear rate over a 60 minute period to twice the normal value coinciding with the gradual development of contracture and coinciding with a cellular K loss and Na gain of about 30 mM/kg. How much of the extra labeled Ca taken up from K-free Ringer's represents a net gain in cellular Ca content is not known. However, evidence has been obtained that some of this labeled Ca enters an intracellular compartment. EDTA in K-free Ringer's solution causes relaxation of ventricles in contracture and also renders the muscle fibers indiscriminately permeable. This indicates that a combination of Ca with sensitive intracellular sites is probably the cause of the K lack contracture.
I N T R O D U C T I O NAn extensive a m o u n t of evidence suggests t h a t calcium ions initiate and regulate contraction in h e a r t muscle (4,7,14), b u t there is c o m p a r a t i v e l y little information a b o u t the h a n d l i n g of calcium ions by h e a r t muscle cells. H o wever, it has been d e m o n s t r a t e d (12, 15) that the a m o u n t of labeled calcium taken up b y frog h e a r t muscle is increased b y omitting potassium f r o m the perfusate and it is well k n o w n t h a t a lack of extracellular potassium, like an excess of calcium, increases the force of contraction. In the present investigation an a t t e m p t has been m a d e to correlate m o r e precisely the contractile effects of potassium deficiency with the cellular m o v e m e n t s of Ca 45, potassium, and sodium in isolated frog h e a r t ventricles.
Zinc depresses the contractile force of electrically driven rat atria logarithmically with time. The threshold concentration is about 5 X 10 -6 M zinc and the half-time for contractile depression at 10 -4 M is about 25 minutes. Zinc also depresses spontaneous activity of atria and alters the transmembrane potential parameters in a manner similar to quinidine. Unlike quinidine, zinc causes an elevation of the resting potential and an elevation of cellular potassium which varies with time in the same way as the resting potential. Exposure to 10 -4 M zinc for 60 minutes causes a statistically significant fall in atrial calcium content and an amount of radioactively labeled zinc is taken up which is quantitatively equal to the calcium lost. Zinc has no effect on rigor caused by iodoacetate but inhibits rigor caused by l-fluoro-2,4 dinitrobenzene. It is postulated that zinc depression of contractile force is not due to metabolic inhibition, probably not due to quinidine-like action on the cell membrane, but may be due to an interference in the handling of calcium by the cell.
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