Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low Km, low Vmax form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high Km, high Vmax form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low Km, low Vmax cyclic AMP-specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted non-specific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells.

Quantification of wear, hemolysis and coagulation deficits in patients with Beall mitral valves.

Fifty-five patients (35 females and 20 males) were studied by noninvasive means 3.5-8.6 years after isolated mitral valve replacement with Models 103 and 104 Beall prostheses. History and physical exams by three physicians, complete hemograms, SMA 18, iron excretion rates, and coagulation profiles were performed. Additionally, electrocardiograms, echocardiograms, phonocardiograms, cardiac series, and high-speed cinefluorography of the prostheses were obtained. Valve wear was assessed by the disc/cage ratio measured from a "three-legged view" with magnification. At a mean duration of 5.85 years after operation, the entire group had a mean disc/cage ratio of operation, the entire group had a mean disc/cage ratio of 0.906 +/- 0.031 vs a normal value of 0.944 +/- 0.014. The group was mildly anemic and had a urinary iron loss that was 40 times normal. The lactic dehydrogenase (LDH) concentration was more than five times normal. The coagulation profiles were abnormal with respect to the bleeding and stypven times, antiheparin activity, fibrin degradation products, and megathrombocyte index. These abnormalities were unrelated to sex, degree of valve wear, and history of thromboembolism. Males were less anemic and had higher urine iron losses than females. Nine patients with severe valve wear (disc/cage ratio less than or equal to 0.87) were significantly more anemic with large urine iron losses and had elevated total bilirubin, serum glutamic oxaloacetic transaminase, and LDH concentrations (ninefold), when compared to nine patients with minimal wear (disc/cage ratio greater than or equal to 0.925). It is emphasized that the findings of a significant anemia, an LDH concentration greater than 1500 mU/ml, and a disc/cage ratio of less than 0.87 in a patient with an isolated Beall mitral valve prosthesis are indicators for the need to replace the prosthesis in the near future. Re-replacement is urged before significant clinical deterioration.

Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations.

Interleukin-8 has been shown by X-ray crystallography and NMR to be a homodimer, suggesting that this is the form which binds to its receptor. Here we measure, for the first time, the monomer-dimer equilibrium of interleukin-8 using analytical ultracentrifugation and titration microcalorimetry and find that it dissociates readily to monomers with an equilibrium dissociation constant of 18 +/- 6 microM at 37 degrees C. The present findings suggest that the monomer is the form which binds to the receptor. Comparison of experimental and structure-based calculated thermodynamics of interleukin-8 dimerization argues for limited subunit conformational changes upon dissociation to monomer.

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography.

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.