Howard Sands

Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle

1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There uas no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.

Cyclic AMP and Protein Kinase in the Spontaneously Hypertensive Rat Aorta and Tissue-Cultured Aortic Smooth Muscle Cells

Cyclic AMP levels and protein kinase activity were determined in the aortas of rats with normotension, moderate and severe spontaneous hypertension. While the cyclic AMP levels were reduced in the aortas from rats with moderate and sever hypertension the protein kinase levels were found to be elevated only in the aortas from rats with severe hypertension. We have grown in tissue culture, aortic smooth muscle cells from the normotensive and severely hypertensive rat. Cultured cells from both strains have similar growth patterns and morphology. The differences seen in cyclic AMP and protein kinase levels in the intact aortas are also seen in the aortic smooth muscle cells in cluture.

Cyclic AMP-stimulated phosphorylation of bovine tracheal smooth muscle contractile and non-contractile proteins

1. Various proteins isolated from bovine tracheal smooth muscle were examined as phosphate acceptor substrates for a cyclic AMP-dependent protein kinase isolated from the same tissue. A fraction prepared in a manner similar to that of skeletal muscle troponin was the best substrate of the presumptive contractile proteins isolate. Actomyosin and tropomyosin were relatively poor substrates. 2. An assay was developed for the rapid detection in a large number of samples of the muscle specific substrate for the protein kinase on which we reported previously. 3. Using this assay, the muscle specific substrate found in bovine tracheal smooth muscle was partially purified resulting in a preparation which when resolved by polyacrylamide gel electrophoresis showed a single peak of 32P incorporated, and which could be further characterized. 4. Our findings suggest that the substrate contains a protein subunit of molecular weight 19 000, which can be phosphorylated at serine and threonine residues, in the presence of cyclic AMP and protein kinase. The phosphate is in a covalent ester linkage with these residues. 5. A phosphoprotein phosphatase was isolated from the bovine tracheal smooth muscle. 6. Bovine tracheal smooth muscle contains cyclic AMP dependent protein kinase and phosphoprotein phospahatase activity as well as the muscle specific substrate, suggesting that these elements may be part of a mechanism which regulates smooth muscle tone.

Actomyosin isolated from bovine tracheal smooth muscle.

Summary A contractile protein (actomyosin) was isolated from bovine tracheal smooth muscle by the use of “classical” procedures. The protein was considered to be actomyosin because it demonstrated: ATPase activity; superprecipitation upon the addition of ATP; and the solubility and extraction characteristics of actomyosin. The ATPase and superprecipitation reactions were not inhibited by EGTA, and did not require calcium. Lack of an effect of either calcium or EGTA could not be reversed by the addition of active bovine skeletal muscle troponin and tropomyosin. No troponin-tropomyosin like activities could be demonstrated in various tracheal muscle fractions.

Effect of Drug Treatment and Aerosoled Antigen Sensitization on Cyclic AMP and β Adrenergic Receptors of Guinea Pig Lung

The interaction between the number of beta adrenergic binding sites and the ability of a beta adrenergic agonist, isoproterenol, to increase cyclic AMP content of guinea pig lung slices was studied. A complex relationship was found. Chronic sensitization of the guinea pig to an aerosol of ovalbumin resulted in lung slices which were hyporesponsive to isoproterenol in vitro, yet possessed an unchanged number of beta adrenergic binding sites. Chronic exposure of guinea pigs to aerosoled isoproterenol or acute treatment with hydrocortisone did not change the number of beta adrenergic binding sites or the responsiveness of the tissue to isoproterenol in vitro. However, chronic hydrocortisone treatment increased the number of binding sites found on the lung slices by 44%, yet there was no change in the responsiveness of the tissue to isoproterenol in vitro. These data suggest that drugs and disease may change the relationship between the various components of the beta adrenergic binding-adenylate cyclase complex of lung.

Phosphorylation of muscle proteins by cyclic adenosine 3',5'-monophosphate-dependent protein kinase from muscle.

Abstract Tissue-specific substrates for a cyclic AMP-dependent protein kinase from bovine tracheal smooth muscle have been partially characterized. The substrates are phosphorylated by purified smooth muscle kinase or by kinases found in crude extracts of bovine smooth, skeletal, and cardiac muscle. The substrates derived from and phosphorylated by smooth muscle protein kinase have a molecular weight of approximately 70 000, as determined by disc gel electrophoresis in the absence of sodium dodecyl sulfate and of approx. 20 000–25 000 when electrophoresed in the presence of sodium dodecyl sulfate. It appears that there are a liminated number of substrates for the smooth muscle cyclic AMP-dependent protein kinase.

Evidence against phosphorylation of a cyclic AMP-dependent protein kinase from bovine tracheobronchial smooth muscle.

Summary The data presented in this report are evidence against the autophos-phorylation of the cyclic AMP-dependent protein kinase isolated from bovine tracheobronchial smooth muscle. This suggests that there may be a fundamental difference in the regulation in vivo of the protein kinases from bovine heart and tracheobronchial smooth muscle. This work was supported by Research Grant HL-14964 from the National Institutes of Health, United States Public Health Service.