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PubMed1995

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Animals↗
PubMed2000

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

Amino Acid Sequence↗
PubMed2001

Receptor phosphorylation mediates estradiol reduction of alpha2-adrenoceptor coupling to G protein in the hypothalamus of female rats.

Estrogen increases evoked norepinephrine release in the hypothalamus of female rodents, in part by reducing the ability of alpha2-adrenoceptors to act as negative feed-back inhibitors of norepinephrine release. Estrogen enhancement of norepinephrine release in the hypothalamus correlates with decreased coupling of the alpha2-adrenoceptor to G protein. To determine the mechanism by which estrogen uncouples alpha2-adrenoceptors from G protein, we tested the hypothesis that estrogen increases alpha2-adrenoceptor phosphorylation. Short-term activation of endogenous serine/threonine phosphatases with protamine or treatment with exogenous phosphatase restored alpha2-adrenoceptor coupling to G protein to control levels in hypothalami from estrogen-exposed female rats. Additional experiments examined whether estrogen alters G protein-coupled receptor kinase expression or activity or serine/threonine phosphatase activity. These proteins are involved in G protein-coupled receptor phosphorylation, internalization, and recycling. Estrogen exposure reduced G protein-coupled receptor kinase mRNA, protein, and activity in the hypothalamus. Furthermore, estrogen treatment reduced serine/threonine phosphatase activity in the hypothalamus. Analysis of ligand binding in subcellular fractions demonstrated that estrogen decreases the fraction of internalized alpha2-adrenoceptors in the hypothalamus.Therefore, estrogen promotes norepinephrine release in the hypothalamus by stabilizing alpha2-adrenoceptor phosphorylation, uncoupling the receptor from G protein. Estrogen may stabilize alpha2-adrenoceptor phosphorylation by inhibiting receptor internalization and dephosphorylation.

Adrenergic alpha-Antagonists↗
PubMed2002

Immediate protamine administration and sheath removal following percutaneous coronary intervention: a prospective study of 429 patients.

We tested the approach of reversing anticoagulation following PCI and immediate sheath removal in 429 consecutive patients. On completion of the PCI, protamine was administered, and the vascular sheath was immediately removed. Stents were used in 364 patients (85%) and GP IIb/IIIa inhibitors were used in 52 patients (12%). Time to achieve hemostasis was 30 +/- 17 min. Minor groin bleeding occurred in six patients. One patient required repair of femoral pseudoaneurysm. Mean creatine kinase at 8 and 16 hr post-PCI were 129 +/- 35 and 145 +/- 32 units, respectively. Creatine kinase rose to > 3 times normal in 12 out of 350 patients (3.4%). Prior to 48 hr, eight patients (1.9%) required emergency PCI or coronary bypass surgery. Follow-up at 30 days observed no deaths and only three target vessel revascularizations (0.7%). In conclusion, immediate reversal of anticoagulation and sheath removal after PCI is safe and feasible.

Aged↗
PubMed1996

Perioperative course and recovery after heparin-coated cardiopulmonary bypass: low-dose versus high-dose heparin management.

OBJECTIVE: To compare two heparin managements for a cardiopulmonary bypass (CPB) procedure with heparin-coated equipment. The hypothesis was that a lower heparin dose may reduce blood loss and homologous transfusion requirements and influence the speed of postoperative recovery. DESIGN: Prospective, randomized, and open study. SETTING: Operating room and intensive care unit in a university hospital. PARTICIPANTS: Twenty-four patients undergoing first-time elective coronary artery surgery. INTERVENTIONS: Heparin-coated CPB equipment (Duraflo II; Baxter-Bentley) was used in all patients. The study group (n = 12) received low-dose (100 IU/kg i.v. and 0 to 1,000 IU/L priming; target level of activated coagulation time [ACT] over 180 seconds during CPB; suction in a red cell washing device); and the control group (n = 12) received high-dose (300 IU/kg i.v. and 5,000 IU/L priming; ACT over 480 seconds; standard cardiotomy suction) heparin management. MEASUREMENTS AND MAIN RESULTS: ACT remained above 200 seconds after the initial heparin dose in the study group for the CPB duration up to 99 minutes. In 11 of 12 patients in the control group, additional heparin was required during CPB. Total doses of heparin and protamine (mean 8,017 v 50,508 IU and 83 v 325 mg, respectively; p < 0.0001), volume of homologous blood transfusion (median 600 v 1450 mL; p < 0.025), and blood products exposure (median 0.5 v 5.0 units/patients; p < 0.05) were significantly lower in the study group. Postoperative chest drainage showed a trend to lower volume loss (median 705 v 930 mL; p < 0.08) in patients managed with low-dose heparin. Oxygenator resistance during CPB, perioperative laboratory analyses (oxygen and metabolic data, hematocrit, platelet count, prothrombin, thrombin, activated partial thromboplastin time, fibrinogen, D-dimers, creatine kinase, and myocardial band of creatine kinase concentration), fluid balance, and the time periods required for extubation, stay in the intensive care unit, and hospital discharge were not different between the groups. There were no evidences of myocardial infarction in any of 24 patients, and all recovered after the procedure. CONCLUSION: Low-dose heparin management enabled uneventful procedures with heparin-coated CPB equipment, significantly decreased protamine and homologous blood requirements, but did not reduce chest drainage or influence the postoperative course and recovery in patients after coronary artery surgery.

Adult↗
PubMed1987

Inhibition of antibodies to CD3 surface antigen and phytohemagglutinin-mediated T cellular responses by inhibiting Ca2+/phospholipid-dependent protein kinase activity with the aid of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride.

Ca2+/phospholipid-dependent kinase activity (C-kinase) plays an important second messenger role in T lymphocyte responses initiated by the cluster of differentiation (CD3) complex and presumably also lectinic receptors. During treatment with submitogenic or mitogenic amounts of phytohemagglutinin, as well as with anti-CD3 monoclonal antibody and 12-O-tetradecanoyl 13-phorbol acetate, the enzyme was intracellularly redistributed between the cytosol and the surface membrane. Submitogenic amounts of lectin and anti-CD3 were ineffective in inducing proliferation unless exogenous interleukin 2 (IL-2) was supplied, implying that even though IL-2 receptors were expressed, additional signals were required for IL-2 production. This would also indicate that there is a direct relationship between activation of C-kinase and expression of IL-2 receptors. The importance of C-kinase was further substantiated by the ability of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H7), a potent inhibitor of this enzyme, to interfere with IL-2 receptor expression and cellular [methyl-3H]thymidine uptake during primary activation. The drug concentration at which these cellular responses were inhibited by 50% was about the same as that which decreased c-kinase activity by 50% in vitro. H7 also prevented anti-CD3-induced translocation in intact cells. This effect may be related to competition with the phosphatidylserine binding site, which is important for membrane attachment. This drug apparently also interferes with the active center of the enzyme as demonstrated by its ability to inhibit Ca2+/phospholipid-independent phosphorylation of protamine sulfate. This additional mode of inhibition may be important in suppressing intact cell responses under circumstances during which the enzyme displacement to the membrane is nonphysiologic in nature, e.g., during treatment with 12-O-tetradecanoyl 13-phorbol acetate.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2003

Calcium sensitivity of a sodium-activated nonselective cation channel in lobster olfactory receptor neurons.

We report that a Na+-activated nonselective cation channel described previously in lobster olfactory neurons, in which phosphoinositide signaling mediates olfactory transduction, can also be activated by Ca2+. Ca2+ activates the channel in the presence of Na+, increasing the open probability of the channel with a K1/2 of 490 nM and a Hill coefficient of 1.3. Ca2+ also increases the sensitivity of the channel to Na+. In some cells, the same channel is Ca2+ insensitive in a cell-specific manner. The nonspecific activator of protein phosphatases, protamine, applied to the intracellular face of patches containing the channel irreversibly eliminates the sensitivity to Ca2+. This effect can be blocked by okadaic acid, a nonspecific blocker of protein phosphatases, and restored by the catalytic subunit of protein kinase A in the presence of MgATP. The Ca2+-sensitive form of the channel is predominantly expressed in the transduction zone of the cells in situ. These findings imply that the Ca2+ sensitivity of the channel, and possibly its regulation by phosphorylation, play a role in olfactory transduction and help tie activation of the channel to the canonical phosphoinositide turnover pathway.

Animals↗
PubMed1994

Significance of electrocardiographic ST elevation during coronary artery bypass surgery.

The aim of this study was to determine the significance of new electrocardiographic (ECG) ST elevation during coronary artery bypass surgery. Multilead ECGs were recorded intraoperatively approximately every 3 min on 105 patients. Cases of new ST elevation were divided into ischemic and those considered to be due to nonischemic causes such as cooling during cardiopulmonary bypass (CPB), defibrillation, new cardiac conduction abnormalities, and pericarditis. The myocardial fraction of creatine kinase (CK-MB) > or = 25 IU/L was considered to be indicative of myocardial injury. Both patients who had ischemic ST elevation prior to CPB and all seven patients who had ST elevation in temporal association with the administration of protamine had peak CK-MB > or = 25 IU/L. One patient with peak CK-MB > or = 25 IU/L did not have ST elevation and was considered to have injury during CPB. Two of these ten patients had Q wave myocardial infarctions (MIs). For the detection of patients with peak CK-MB > or = 25 IU/L, the sensitivity of ischemic ST elevation was 90% and the specificity was 100%. A history of MI prior to surgery and a history of Type I diabetes were associated with peak CK-MB > or = 25 IU/L (P < 0.05).

Aged↗
PubMed2000

Two different mechanisms for activation of cyclic PIP synthase: by a G protein or by protein tyrosine phosphorylation.

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation.

Adenosine Triphosphate↗
PubMed1980

Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.

Animals↗