Effect of cesium ions on the electrotropic actions of acetylcholine in the rabbit atrium

Cs+ (15-20 mM) decreases the electrotropic vagal effects on an isolated vagal innervated rabbit atrium. By means of investigating the action potentials and the phase plane trajectories of trabeculae from the rabbit atrium using a modified single sucrose gap technique the anomalous rectification disappears and also the effect of acetylcholine (ACh) on action potential duration. We presume that the anomalous rectification should be a necessary condition of electrotropic vagal (ACh) action on the rabbit atrium.     

Disparity between expression of transferrin receptor ligand binding and non-ligand binding domains on human lymphocytes

We compared transferrin receptor (TfR) expression on human peripheral blood lymphocytes (PBL) activated by phorbol myristate acetate (PMA) or L-phytohemagglutinin (LPHA) using two techniques: (1) 125I-iron-saturated transferrin (FeTf) binding, (2) reactivity with monoclonal anti-TfR antibodies--OKT9 and B3/25. These monoclonal antibodies do not block FeTf binding, and therefore bind to TfR domains separate from the ligand binding site. Unstimulated PBL bound fewer than 1,000 molecules of 125I-FeTf per cell, and less than 5% of cells expressed TfR antigens detected by OKT9 or B3/25. 125I-FeTf binding and antibody binding increased in parallel on LPHA-activated PBL. After exposure to LPHA for 72 hr, 125I-FeTf binding increased 100-fold to 10(5) molecules per cell and greater than 50% of cells expressed TfR antigens. By contrast, PMA activation of PBL markedly increased binding of OKT9 and B3/25 but not the binding of 125I-FeTf. Cell surface expression of TfR antigens seen by OKT9 and B3/25 did not differ between LPHA- and PMA-activated PBL. However, after 72 hr with PMA, 125I-FeTf binding increased only 6-fold and consistently remained at less than 10(4) molecules per cell. Therefore, PMA induced a disparity between expression of TfR ligand binding domains and immunological domains at the cell surface. Cell proliferation assessed by fluorescent DNA analysis was similar in cultures stimulated by LPHA or PMA. These data indicate that lymphoid cells may possess a mechanism for modulating TfR expression in which down-regulation of FeTf binding occurs without receptor internalization. Alternatively, it is possible that this observation may reflect a membrane perturbation effect of PMA.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.     

Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium

Effects of the plant alkaloid Aconitine on the kinetics of sodium channels were studied in enzymatically isolated single cells of the mouse ventricular myocardium. Aconitine (1 mumol/l) induced a prolongation of the 90% repolarization of action potentials from 52.4 +/- 3.7 ms to 217.0 +/- 12.5 ms. Delayed terminal repolarization and oscillatory afterpotentials preceded spontaneous activity with high frequencies. Peak sodium currents were diminished from 28.0 +/- 9.0 to 14.0 +/- 6.0 nA. The reversal potential of the sodium current was shifted from 16.0 +/- 11.0 to -8.0 +/- 6.0 mV (52.5 mmol/l extracellular sodium concentration) suggesting a decreased selectivity of the Aconitine-modified Na channels. The m-affinity-curves were shifted 31 mV towards more negative potentials at a constant slope. The h affinity-curves were shifted in the same direction by 13 mV. The slope parameter of the h affinity-voltage relationship was enlarged from 9.1 +/- 2.2 mV to 15.6 +/- 4.4 mV. Shifts in m affinity and h affinity resulted in an increased "window". The alkaloid modified channels inactivated extremely slowly at potentials negative to -40 mV, but showed a fast and complete inactivation at potentials positive to -40 mV.     

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH.

The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70. This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra. Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies. The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH. Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90. Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH. However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.     

Potassium dependence for sperm-egg fusion in mice

In this study, we examined the potassium requirements for sperm-egg fusion in mouse. Zona-free mouse eggs prepared by the method described by Boldt and Wolf were inseminated with capacitated sperm in culture media containing 0-6 mM extracellular K+, and scored for penetration. Penetration of zona-free eggs was dependent on extracellular K+, with no penetration observed under K(+)-free conditions. Media transfer experiments indicated that the lack of penetration observed was due to effects on fusion, and not on postpenetration events such as sperm head decondensation. To analyze whether the K+ effect was attributable to an effect on the sperm (i.e., occurrence of acrosome reactions), sperm were treated with the Ca2+ ionophore A23187 before insemination. Less than 5% of zona-free eggs were penetrated with ionophore-treated sperm under K(+)-free conditions, suggesting that K+ is required for fusion per se. Addition of ionophore to insemination cultures similarly did not overcome the block to fusion observed under K(+)-free conditions. The potassium channel blockers 4-aminopyridine (0.1-5 mM) and tetraethyl ammonium chloride (5-50 mM) had no inhibitory effect on fusion. These data indicate that extracellular K+ is required for sperm-egg fusion and that this requirement may not involve membrane K+ channels.     

Repression of the Plastidic Isoenzymes of Aldolase, 3-Phosphoglycerate Kinase, and Triosephosphate Isomerase in the Barley Mutant "albostrians"

White leaves of the mutant line albostrians and green leaves of the wild-type cultivar Salome of barley (Hordeum vulgare L.) were screened for the presence of plastidic and cytosolic isoenzymes of sugar-phosphate metabolism. Isoenzyme separation was achieved by anion-exchange chromatography on Fractogel TSK DEAE-650(S). The mutant tissue had a markedly reduced level of plastidic 3-phosphoglycerate kinase, triosephosphate isomerase, and aldolase activity. In contrast, the activity of plastidic glucosephosphate isomerase, fructose 1,6-bisphosphatase, 6-phosphogluconate dehydrogenase, starch phosphorylase, and ADP-glucose pyrophosphorylase was in the same range as in wild-type leaf tissue. The activity of the corresponding cytosolic isoenzymes (including UDP-glucose pyrophosphorylase) showed essentially no differences in mutant and wild type. The same trend was observed in dark-grown mutant and wild-type leaves. Interestingly, the total activity levels of all isoenzymes were about the same when comparing dark-grown and light-grown mutant or wild-type plants. From these data, it is concluded that mutant leaves exhibit a selective decrease of a subgroup of plastidic isoenzymes associated with the Calvin cycle.