Phosphodiesterase 4 interacts with the 5-HT4(b) receptor to regulate cAMP signaling.

Phosphodiesterase (PDE) 3 and PDE4, which degrade cyclic adenosine monophosphate (cAMP), are important regulators of 5-hydroxytryptamine (5-HT) 4 receptor signaling in cardiac tissue. Therefore, we investigated whether they interact with the 5-HT4(b) receptor, and whether A-kinase anchoring proteins (AKAPs), scaffolding proteins that bind to the regulatory subunit of protein kinase A (PKA) and contribute to the spacial-temporal control of cAMP signaling, are involved in the regulation of 5-HT4(b) receptor signaling. By measuring PKA activity in the absence and presence of PDE3 and PDE4 inhibitiors, we found that constitutive signaling of the overexpressed HA-tagged 5-HT4(b) receptor in HEK293 cells is regulated predominantly by PDE4, with a secondary role for PDE3 that is unmasked in the presence of PDE4 inhibition. Overexpressed PDE4D3 and PDE3A1, and to a smaller extent PDE4D5 co-immunoprecipitate constitutively with the 5-HT4(b) receptor. PDE activity measurements in immunoprecipitates of the 5-HT4(b) receptor confirm the association of PDE4D3 with the receptor and provide evidence that the activity of this PDE may be increased upon receptor stimulation with 5-HT. A possible involvement of AKAPs in 5-HT4(b) receptor signaling was uncovered in experiments using the St-Ht31 inhibitor peptide, which disrupts the interaction of AKAPs with PKA. However, St-Ht31 did not influence 5-HT4(b) receptor-stimulated PKA activity, and endogenous AKAP79 and gravin were not found in immunoprecipitates of the 5-HT4(b) receptor. In conclusion, we found that both PDE3A1 and PDE4D3 are integrated into complexes that contain the 5-HT4(b) receptor and may thereby regulate 5-HT4(b) receptor-mediated signaling.

Utility of long-term surveillance endomyocardial biopsy: a multi-institutional analysis.

BACKGROUND: The utility of long-term endomyocardial biopsy surveillance in heart transplant recipients has been questioned. This study was undertaken to identify risk factors for late rejection and to examine the impact of different biopsy surveillance protocols on outcomes using the registry of the Cardiac Transplant Research Database. METHODS: The study group consisted of all adult patients who underwent heart transplantation at the 33 centers participating in this investigation between January 1, 1993 and January 1, 2002, survived past the second post-transplant year, and were followed-up by a defined surveillance biopsy protocol. RESULTS: During a follow-up that consisted of 24,137 patient-years, 1,626 late rejections occurred. Shorter time since transplant, history of rejection, younger age and African-American ethnicity of the recipient were strong risk factors for late rejection. The practice of surveillance biopsy varied among institutions. Continued surveillance increased the rate of diagnosis of late rejection (RR = 1.3, p = 0.002). There was no reduction in the incidence of hemodynamically compromising rejection and no increase in survival in patients with long-term vs intermediate-term surveillance. Short-term surveillance was associated with an increased incidence of hemodynamically compromising rejection, particularly among high-risk patients, and increased mortality in African-American patients. CONCLUSIONS: There are no apparent benefits from surveillance biopsy beyond 5 years post-transplant. Surveillance biopsy between 2 and 5 years post-transplant was found to reduce mortality in African-American recipients. Non-African-American recipients at high risk for late rejection will likely benefit from surveillance up to 5 years post-transplant.

Identification of a novel isoform of the cyclic-nucleotide phosphodiesterase PDE3A expressed in vascular smooth-muscle myocytes.

We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.

Therapeutic potential of cyclic nucleotide phosphodiesterase inhibitors in heart failure.

There are several reasons to believe that agents that augment cAMP-mediated signalling in cardiac myocytes should have beneficial effects in patients with heart failure. However, clinical trials of first-generation cyclic nucleotide phosphodiesterase (PDE3) inhibitors, which raise cAMP content by blocking its hydrolysis, have shown that chronic administration of these drugs affect survival adversely. The problem may be the non-selective activation of a broad spectrum of cAMP-regulated cellular responses these agents elicit. More selective (or alternatively selective) cyclic nucleotide PDE inhibitors might improve results by evoking a more restricted set of cellular responses.

[The effect of live tularemia vaccination on the short- and long-term results of combined therapy of patients with endometrial cancer]. / Vliianie tuliaremiinoi zhivoi vaktsiny na blizhaishie i otdalënnye rezul'taty kombinirovannogo lecheniia bol'nykh rakom tela matki.

A randomized investigation of the immediate (wound healing) and end-results (relapse and metastasis frequency, recurrence-free period, 2-, 3- or 5-year survival, lethality rate and mean lifespan) of combined (surgery + distant gamma-therapy) treatment of two groups of patients with endometrial carcinoma was carried out. The study group was operated on days 15-20 after immunization with tularemia live vaccine (TLV) while the controls were had no preliminary immunization. It was shown that all indices of the immediate and end-results of treatment were significantly better in the study group for all stages. It is concluded that TLV immunization given 15-20 days before surgery is followed by a lower frequency of postoperative complications and better end-results.

Beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors: shifting the focus from inotropy to cyclic adenosine monophosphate.

Clinical trials of beta-adrenergic receptor agonists and cyclic nucleotide phosphodiesterase inhibitors in heart failure have demonstrated a reduction in survival in treated patients despite initial inotropic responses. These findings have led many to infer that activation of the mechanisms through which contractility is increased has deleterious effects on failing myocardium. It should be remembered, however, that these agents act proximately by raising intracellular cyclic adenosine monophosphate (cAMP) content and stimulating protein phosphorylation by cAMP-dependent protein kinase, and that the proteins whose phosphorylation contributes to the inotropic responses may be different from the proteins whose phosphorylation contributes to the reduction in survival. Evidence in support of the latter interpretation is presented, and potential therapeutic approaches through which the phosphorylation of different proteins might be selectively affected are considered.

Identification and quantitation of cAMP-dependent protein kinase R subunit isoforms in subcellular fractions of failing human myocardium.

Isoforms of regulatory (R) subunits of cAMP-dependent protein kinase were identified immunochemically and quantified in soluble and washed particulate fractions of failing human left ventricular myocardium. The predominant isoforms in both fractions were RI alpha and RII alpha. Both isoforms were present in comparable amounts in these fractions, although RII alpha subunits were somewhat more prevalent than RI alpha subunits in washed particulate fractions. The ratio of R subunits to catalytic (C) subunits was three-fold higher in soluble than in particulate fractions. Identical observations were made in preparations from non-failing human left ventricular myocardium. Since RI and RII have different affinities for cAMP and may direct catalytic activity to different substrates, the presence of both subunits in both soluble and particulate fractions provides a mechanism whereby the compartment-selective changes in cAMP content that have been described in failing human myocardium may affect not only the level but also the profile of protein phosphorylation in these compartments. The high R:C subunit ratio in soluble fractions suggests that cytosolic kinase activity in human myocardium may be less sensitive to changes in cAMP content than membrane-bound kinase activity, and this may contribute to the different effects of increases in soluble and particulate cAMP content on intracellular Ca2+transients and contraction and relaxation.

[The effect of tularemia live vaccine on contrasuppression of lymphocytes in patients with cancer of the corpus uteri during combined therapy and long term]. / Vliianie tuliaremiinoi zhivoi vaktsiny na èffekt kontrasupressii limfotsitov krovi u bol'nykh rakom tela matki v protcesse kombinirovannogo lecheniia i v otdalënnye sroki.

A significant decrease in the percentage of women showing contrasuppression of blood lymphocytes, as compared with control, was identified (p < 0.1); it was particularly low at terminal stages (III-IV). Surgery and subsequent radiotherapy were followed by further inhibition, particularly in terminally-ill patients. Two or three years after treatment, the numbers of patients in all stage groups showing contrasuppression increased, reaching the initial, i.e. lower than normal, levels. However, contrasuppression indices rose to normal in patients immunized with tularemia live vaccine (p > 0.05). This effect was observed in patients of all stage groups, immunized prior to treatment, on days 15-20 after immunization. Normal levels of contrasuppression were maintained during treatment and 2-3 years on. Both contrasuppression level and stage of tumor may serve as a criterion of gravity of disease and prognosis.

[Effect of live tularemia vaccine on some parameters of immune and non-specific antitumor resistance in endometrial cancer patients during combined therapy and later]. / Vliianie tuliaremiinoi zhivoi vaktsiny na dinamiku nekotorykh pokazatelei immunoi i nespetsificheskoi protivoopukholevoi rezistentnosti organizma bol'nykh rakom tela matki v protsesse kombinirovannogo lecheniia i v otdalënnye sroki.

The levels of T-system lymphocytes, macrophageal transformation of mononuclears and phagocytic activity of blood neutrophils were assayed in 194 patients with endometrial tumors, stage I-IV, (FIGO, 1988), 10-15 days after surgery, a subsequent course of telegammatherapy and 2-3 years after treatment. Ninety-seven patients of the study group were challenged by immunization with a live tularemia vaccine (LTV) 15-20 days before surgery, while the same number of controls were not. The study was randomized. Pre-operative LTV immunization showed an immunoprotective effect at all stages of combined treatment (surgery + telegammatherapy) given for endometrial carcinoma. Moreover, all said indices came back to normal in the study group 2-3 years after treatment. After surgery alone (standard uterine extirpation or with adnexa), controls with malignancies at the same stages revealed a significant decrease in the indices which fell further after radiotherapy. In the latter group, the indices came back to initial values which were below the norm.

cAMP-mediated signal transduction and sarcoplasmic reticulum function in heart failure.

There is evidence that the effects of beta-adrenergic receptor agonists on myocardial contractility result principally from the phosphorylation of phospholamban by cAMP-dependent protein kinase and the consequent deinhibition of SERCA2 activity and stimulation of sarcoplasmic reticulum Ca2+ transport. An impairment in beta-adrenergic receptor-stimulated cAMP generation, attributable to down-regulation of beta 1-adrenergic receptors and increased activity of G alpha i and G protein-coupled receptor kinase, has long been recognized in failing human myocardium. This impairment is associated with a compartment-specific decrease in sarcoplasmic reticulum cAMP content that may selectively reduce phospholamban phosphorylation. Published and preliminary results indicate that two plausible explanations for this compartment-specific decrease--a reduction in sarcoplasmic reticulum-associated cAMP-dependent protein kinase or an increase in sarcoplasmic reticulum-associated cAMP phosphodiesterase--are unlikely. Instead, there is reason to believe that the selective reduction in beta 1-adrenergic receptor density in failing myocardium is causally related to this compartment-specific decrease in cAMP content through an as-yet-undetermined mechanism. The fact that the modulation of SERCA2 activity by phospholamban is preserved in failing human myocardium offers an opportunity for improvement in the therapy of heart failure.

[Immediate and long-term results of combined therapy of patients with endometrial neoplasms depending on the effect of counter-suppression of blood lymphocytes]. / Blizhaishie i otdalënnye rezul'taty kombinirovannogo lecheniia bol'nykh rakom tela matki v zavisimosti ot éffekta kontrasupressii limfotsitov krovi.

The short-term (wound healing) and end-results (relapse and metastasis frequency, duration of relapse-free survival and 2-, 3- and 5-year survival) of combined treatment (surgery + gamma therapy) of 97 patients with endometrial carcinoma (stage I-II and stage III-IV) (FIGO, 1988) versus the effect of preoperative counter-suppression of blood lymphocytes were investigated. Both short-term and end-results appeared to be much better in patients showing the counter-suppression effect; they were much worse in patients who revealed the effect prior to combined therapy, and still worse--in cases of inverse effect involving enhanced suppression. It is suggested that the effect of blood lymphocyte counter-suppression before combined treatment is a factor of favorable prognosis in patients with endometrial carcinoma.

[Adjuvant immunotherapy for patients with cancer of corpus uteri]. / Spocob ad'iuvantnoi immunoterapii bol'nykh rakom tela matki.

A randomized study was carried out of the efficacy of a new procedure of adjuvant immunotherapy for cancer of corpus uteri in conjunction with anticoagulant treatment. Ninety-seven patients (76-stage I-II and 21-stage III-IV) were inoculated tularemia booster (TB) and received fibrinolysin, pelentan and aspirin 15-20 days before combined treatment including uterine extirpation or extirpation with adnexa followed by telegammatherapy. Two groups of identical numbers of patients with an identical distribution of tumor progression stage were used as controls. No vaccination was carried out in one of them while TB inoculation was performed without anticoagulants in the other. A higher effectiveness of immunostimulation accompanied by anticoagulant administration was registered on the basis of the short-term (wound healing) and end results (relapse and metastasis frequency, duration of relapse-free survival and 2-, 3- and 5-year survival).

Expression and activity of low Km, cGMP-inhibited cAMP phosphodiesterase in cardiac and skeletal muscle.

The expression and activity of low Km, cGMP-inhibited cAMP phosphodiesterase (PDE3)4 were examined in rabbit and canine cardiac and skeletal muscle. In cardiac muscle, a cDNA probe whose sequence encompasses the catalytic domain of human myocardial PDE3 (PDE3A) hybridized predominantly with a 7.2-7.4 kb mRNA. No hybridization was observed in preparations from slow or fast twitch skeletal muscle. Likewise, PDE3 activity was present in cytosolic and microsomal fractions of cardiac muscle but was absent from cytosolic and microsomal fractions of slow twitch and fast twitch skeletal muscle. These results, which demonstrate the absence of PDE3 from slow and fast twitch mammalian skeletal muscle, further delineate the differences in beta-adrenergic receptor-mediated signal transduction pathways in cardiac and skeletal muscle.

Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells.

cDNAs encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (H) cDNA libraries. The deduced amino acid sequences of RcGIP1 and HcGIP2 are very similar in their conserved catalytic domains but differ in their N-terminal regulatory domains [Meacci, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., et al. (1993) J. Biol. Chem. 268, 18573-18579]. cDNAs encoding both rat adipocyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncated forms lacking much of the putative N-terminal domain) were expressed in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant proteins exhibited the expected subunit molecular mass, immunologic reactivities, and characteristics of native membrane-associated forms of the enzymes, e.g., high affinity for cAMP (Km), sensitivity to the selective cGI PDE inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-length recombinants were predominantly particulate, whereas the truncated HcGIP2 forms were cytosolic suggesting that N-terminal domains contain structural determinants important for membrane association. Both fibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated in vitro by cAMP-dependent protein kinase; tryptic [32P]peptides released from rat adipocyte 32P-cGI PDE and 32P-RcGIP1 exhibited identical electrophoretic profiles suggesting that the same peptides are phosphorylated in both.

[Evaluation of immunotherapy in breast cancer (stage III-IV) and in cancer of the corpus uteri (stage I-III)]. / Otsenka rezul'tatov immunokhimioterapii bol'nykh rakom molochnoi zhelezy (III-IV stadiia) i tela matki (I-III stadii).

A new clinically-tested procedure of adjuvant immunotherapy carried out in conjunction with anticoagulant treatment is discussed. Application of said method has significantly improved the short-term and end results of treatment of inoperable cases of breast cancer, stage III-IV, receiving chemotherapy and patients with cancer of corpus uteri, stage I-III, receiving combined therapy plus radiation.

Pharmacologic and hemodynamic effects of combined beta-agonist stimulation and phosphodiesterase inhibition in the failing human heart.

STUDY OBJECTIVES: We measured the individual and combined effects of the beta-agonist dobutamine and the phosphodiesterase inhibitor enoximone both in vitro and in vivo in the failing human heart. DESIGN: This was an unblinded, prospective study. SETTING AND PATIENTS: The in vitro measurements were performed on 20 hearts obtained from subjects with end-stage biventricular failure and from seven normal hearts. The in vivo measurements were performed in eight subjects with class IV heart failure. INTERVENTIONS AND MEASUREMENTS: The in vitro measurements of enoximone, dobutamine, and the combination of these agents were phosphodiesterase activity using a sarcoplasmic reticulum-enriched preparation, cyclic adenosine monophosphate (cAMP) accumulation using particulate fractions, and tension response using isolated right ventricular trabeculae. The dose response to dobutamine, the combination of enoximone and dobutamine, and the combination of nitroprusside and dobutamine were measured in vivo using invasive hemodynamic monitoring. RESULTS: In vitro, enoximone exhibited dose-dependent inhibition of phosphodiesterase activity. The addition of enoximone to dobutamine resulted in an upward and leftward shift of the dobutamine dose-response curve for both cAMP production and contractile response. In vivo, enoximone significantly shifted the dobutamine dose-response curves for cardiac index, left ventricular stroke work index, and heart rate upward and to the left; and shifted the dobutamine dose-response curves for right atrial, pulmonary arterial, and pulmonary wedge pressures downward and to the right. CONCLUSIONS: Enoximone exerts favorable effects on cardiac performance that are additive to those produced by dobutamine. These effects are mediated by increasing cellular cAMP concentrations through independent, additive mechanisms.

Effect of ryanodine on sarcoplasmic reticulum Ca2+ accumulation in nonfailing and failing human myocardium.

BACKGROUND: The purpose of this study was to determine whether abnormal Ca2+ release through ryanodine-sensitive Ca2+ channels in the sarcoplasmic reticulum might contribute to the abnormal [Ca2+]i homeostasis that has been described in failing human myocardium. METHODS AND RESULTS: Occupancy of low-affinity ryanodine binding sites on ryanodine-sensitive Ca2+ channels stimulates oxalate-supported, ATP-dependent Ca2+ accumulation in sarcoplasmic reticulum-derived microsomes by inhibiting concurrent Ca2+ efflux through these channels. We examined the effects of 0.5 mmol/L ryanodine on 45Ca2+ accumulation in microsomes prepared from nonfailing (n = 8) and failing (n = 10) human left ventricular myocardium. In the absence of ryanodine, 45Ca2+ accumulation reached similar levels in microsomes from nonfailing and failing hearts. Incubation with 0.5 mmol/L ryanodine caused a 52.2 +/- 6.5% increase in peak 45Ca2+ accumulation in microsomes from nonfailing hearts and a 24.3 +/- 4.1% increase in microsomes from failing hearts. The density of high-affinity ryanodine binding sites and the inhibition of [3H]ryanodine dissociation from these sites by 0.1 mmol/L ryanodine were similar in microsomes from nonfailing and failing hearts. CONCLUSIONS: These results, which demonstrate a diminished stimulation of Ca2+ accumulation by ryanodine in sarcoplasmic reticulum-derived microsomes from failing human myocardium that could be explained by an uncoupling of the occupancy of low-affinity ryanodine binding sites from the reduction in the open probability of these channels or by concurrent Ca2+ efflux through a ryanodine-insensitive mechanism, are evidence that increased efflux of Ca2+ from the sarcoplasmic reticulum may contribute to the abnormal [Ca2+]i homeostasis described in failing human myocardium.

[The effect of tularemia vaccine on some parameters of immune and non-specific anti-tumor resistance in patients with endometrial tumors]. / Vliianie tuliaremiinoi vaktsiny na nekotorye pokazateli immunoi i nespetsificheskoi protivoopukholevoi rezistentnosti organizma bol'nykh rakom tela matki.

On days 15-20 after immunization of patients suffering from endometrial tumors with tularemia live vaccine, a significant increase was registered in T-cell population of the immune system, macrophagal transformation of mononuclears and phagocyticity of blood neutrophiles which are generally reduced in stages III-IV. The study group included cases of all tumor stages. Said indices reached normal levels in stage I-II only.