Caldesmon is present in human and pig erythrocytes

Caldesmon, a major actin- and calcium-dependent calmodulin-binding protein, is now considered as an essential inhibitory factor of the actomyosin machinery in smooth muscle cells as well as in non-muscle cells. Since its structure seems to vary with the cell in a same species and because platelet and erythrocyte have a common embryonic origin, we have used the affinity purified antibody raised against the platelet caldesmon to determine whether this protein is present in erythrocyte. Using the immunoblotting technique, we show here that, in whole erythrocytes, only a single polypeptide crossreacts with this polyclonal antibody. A 71-72 kDa Mr has been calculated from SDS-PAGE. It is therefore different from those of the gizzard (Mr 145-150 kDa) or the platelet (Mr 80 kDa) proteins. Furthermore, we also give evidence that it is not adducin since this newly discovered calcium-dependent calmodulin-binding protein of erythrocyte, does not crossreact with the polyclonal affinity purified antibody raised against platelet caldesmon.     

The tyrosine kinase inhibitor genistein increases basal cAMP and potentiates forskolin-induced cAMP accumulation in A549 human airway epithelial cells

Genistein is often used as an inhibitor of tyrosine kinases. A less studied side effect of genistein is an inhibition of cyclic AMP-phosphodiesterase (cAMP-PDE) activity resulting in increased cAMP accumulation. The effect of genistein on intracellular cAMP-levels, basal and forskolin-induced, was studied in A549 human airway epithelial cells and compared with the unspecific PDE inhibitor, isobutylmethylxanthine (IBMX). It was shown that genistein (50 M) increased basal cAMP and potentiated forskolin-induced cAMP accumulation to the same extent as IBMX (100 M). Thus, the use of genistein in studies on signaling transductions may result in erroneous conclusions since increased cAMP may cause or contribute to the observed effects.     

An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.     

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine stimulates reproduction in rabbit females

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate and cyclic guanosine monophosphate phosphodiesterases, on the reproductive efficiency of gonadotropin-stimulated rabbits (Oryctolagus cuniculus, Leporidae, Lagomorpha). The ovarian cycle and ovulation of control rabbits was induced by pregnant mare serum gonadotropin (PMSG) followed by administration of human chorionic gonadotropin (hCG; first series of experiments) or gonadotropin-releasing hormone (GnRH; second series of experiments). Experimental animals received PMSG and hCG together with IBMX (at 5 or 25 μg/animal) or GnRH together with IBMX (at 50 or 500 μg/animal). After ovulation and mating, in the first series of experiments, animals were killed; the pronuclear-stage eggs were flushed from the oviducts and cultured up to blastocyst cell stage. Numbers of ovarian corpora lutea, ovulated oocytes, and oocyte-derived embryos reaching blastocyst stage were determined. In the second series of experiments, all the animals were kept until parturition, when the pregnancy and birth rate, litter size, and number, viability, and body weight of pups were recorded. IBMX injections at doses of 5 or 25 μg/animal significantly increased the number of ovulations/corpora lutea, harvested zygotes, and embryos derived from these zygotes. Administration of IBMX at doses of 500 μg/animal or 50 μg/animal to nulliparous young animals (4.5 mo of age) significantly increased their pregnancy rate and birth rate, litter size, and litter weight. In multiparous old animals (2 yr of age), IBMX at a dose of 50 μg/animal, but not 500 μg/animal, significantly increased their pregnancy rate and litter size, but not the birth rate, number of pups per female, or litter weight. These data demonstrate that (1) IBMX can enhance the stimulatory effect of GnRH/gonadotropins on rabbit ovulation, oocyte maturation, embryo yield and development, pregnancy and birth rate, and number, viability, and body weight of pups; (2) nulliparous young females (4.5 mo of age) are more sensitive to IBMX treatments than the multiparous old animals (2 yr of age); and (3) cyclic nucleotides-dependent intracellular mechanisms are involved in control of rabbit reproductive functions and IBMX, an activator of these mechanisms, can be a stimulator of reproduction and fertility.     

Gap junction-dependent increases in smooth muscle cAMP underpin the EDHF phenomenon in rabbit arteries

We have investigated the role of cAMP in nitric oxide (NO)- and prostanoid-independent vascular relaxations evoked by acetylcholine (ACh) in isolated arteries and perfused ear preparations from the rabbit. These EDHF-type responses are shown to be associated with elevated cAMP levels specifically in smooth muscle and are attenuated by blocking adenylyl cyclase or protein kinase A (PKA). Relaxations are amplified by 3-isobutyl-1-methylxanthine, which prevents cAMP hydrolysis, while remaining susceptible to inhibition by the combination of two K(Ca) channel blockers, apamin and charybdotoxin. Analogous endothelium- and cAMP-dependent relaxations were evoked by cyclopiazonic acid (CPA) which stimulates Ca(2+) influx via channels linked to the depletion of Ca(2+) stores. Responses to ACh and CPA were both inhibited by interrupting cell-to-cell coupling via gap junctions with 18alpha-glycyrrhetinic acid and a connexin-specific Gap 27 peptide. The findings suggest that EDHF-type responses are initiated by capacitative Ca(2+) influx into the endothelium and propagated by direct intercellular communication to effect relaxation via cAMP/PKA-dependent phosphorylation events in smooth muscle.     

Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.     

Glucose transport inhibition in human erythrocytes by calmodulin antagonists

Calmodulin antagonists (tryphtazin, lidocaine, dykain, palmitate) inhibit glucose transport from human erythrocytes. Glucose efflux inhibition is proportional to the concentration of antagonists in the medium and is of uncompetitive character. It is accompanied by a decrease in the maximum transport rate with the unchanged constant of dissociation in the complex: carrier-sugar. Calcium ionophores A23187 and divaleryldibenzo-18-crown-6 eliminated the inhibiting effect of pharmacological agents on glucose transport. The authors think that the glucose transport inhibition under the influence of calmodulin antagonists may be realized through the calmodulin-dependent chain inhibition under the influence of calmodulin antagonists in the carbohydrate transport system.     

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.     

Comparative distribution of glyceraldehyde 3-phosphate dehydrogenase activity in human, guinea-pig, rabbit and mouse erythrocytes

1. Guinea-pig erythrocytes contain half the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD) present in human cells. About 60% of their total activity is membrane-bound. 2. Rabbit erythrocytes also contain half the amount of the enzyme of human red cells. The distribution of G3PD in rabbit cells, however, is similar to that of human cells with 70% of the enzyme being membrane-bound. 3. Mouse erythrocytes contain about two-thirds of G3PD activity present in human cells. All their enzyme activity is present in membrane-free hemolysate. 4. Non-human erythrocyte membrane proteins, in addition, have relatively greater amount of band 2.1, lack band 2.2, have a more heterogenous band 3 than its human counterpart, and have overlapping bands 4.1 and 4.2.     

Ketotifen increases cyclic adenosine 3',5'-monophosphate in intact human lymphocyte and potentiates other adenylate cyclase activating agents

We have investigated the effects of ketotifen on the cyclic adenosine 3',5'-monophosphate (cyclic AMP) response of intact human lymphocyte and its interaction with adenylate cyclase activating agents. In the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine), ketotifen (10(-8)-10(-4) M) caused an 80% increase in cyclic AMP content of human lymphocyte, a magnitude similar to that observed with hydrocortisone. The cyclic AMP level peaked at about 15 minutes and remained elevated for at least 45 minutes. In addition, ketotifen (10(-6)-10(-4) M) markedly potentiated the effect of several adenylate cyclase stimulating agents, including L-isoproterenol, prostaglandin E1 and forskolin. The biochemical mechanisms underlying these effects are unknown. It may be at least partly related to the ability of ketotifen to reverse and prevent beta 2 adrenoceptor desensitization and to promote the formation of hormone - nucleotide - high affinity receptor complex. These effects may contribute to its prophylactic effect in the treatment of bronchial asthma.     

Bioassays of APONIN-3 and -4 with rabbit erythrocytes

Rabbit erythrocytes in methanolic phosphate medium were used to bioassay the activity of authentic samples of methyl stearate and methyl palmitate (in 10% methanol:90% water, v/v), which had been identified as apparent oceanic naturally occurring cytolins (APONIN-3 and -4) produced by Nannochloris oculata. The two natural products are notable for cytolytic activity toward the unarmoured dinoflagellate, Gymnodinium breve Davis, an organism responsible for red tides consisting of harmful algal blooms in the Gulf of Mexico and along the eastern coast of the United States. Bioassays were done with heparinized rabbit blood. The absorbance at 540 nm was observed for 15 min in comparison with a sample treated with haemolysing agent. The results indicated that at reasonable concentrations of 1-10 ppm, neither was a haemolysin, although such concentrations caused cytolysis of G. breve cultures.     

Comparison of photodynamic effect with respect to human and rabbit erythrocytes

Parameters of photoinduced lysis are studied in human and rabbit erythrocytes (photosensitizer-Radachlorine, the light source Shatl, λ = 633 nm). The higher sensitivity to irradiation is revealed in rabbit erythrocytes. Treatment of erythrocytes with trypsin showed the surface proteins in human cells to produce a protective action. Trypspnization of rabbit erythrocytes produced the opposite effect-the rate of photohemolysis increased. Results of the study indicate the differences in sensitivity to the photoinduced lysis of erythrocytes of different species and participation of erythrocyte proteins in the effect of photohemolysis.