Depolarization-induced 86Rb+ efflux in CHO cells expressing a recombinant potassium channel.

Cells expressing a recombinant human voltage-activated potassium channel (K-channel), Kv1.5, have been used in a functional assay that measures depolarization-stimulated 86Rb+ efflux as an indicator of K-channel function. Neither untransfected nor vector-transfected cells display measurable 86Rb+ efflux under depolarizing conditions. The depolarization-induced 86Rb+ efflux is blocked by standard K-channel blockers quinine, 4-aminopyridine and 3,4-diaminopyridine, but not by tetraethylammonium, quinidine, glibenclamide, or several peptide toxins. The pharmacological profile of the recombinant system reflects that reported for the channel in its native state. In such a system with no observable endogenous background, analysis of recombinant K-channel subtypes allows rapid assessment of pharmacological agents with isoform selectivity and specificity. Inclusion of compounds of unknown activity in an assay such as this could identify agents capable of modulating specific K-channel isoforms. Development of this high through-put assay system for the study of specific isoforms is a critical step in the identification and development of drugs that affect the desired target tissues with predictable pharmacology and minimal side effects due to nonselective K-channel interaction.

5-HT3 receptor-independent inhibition of the depolarization-induced 86Rb efflux from human neuroblastoma cells, TE671, by ondansetron.

The 5-HT3-receptor antagonist, ondansetron, has been shown to have positive effects in selected in-vivo models of memory impairment and anxiety. The exact mechanisms underlying such bioactivities are unknown. In the present work, an 86Rb efflux bioassay was used to show that ondansetron has a unique ability to block voltage-gated potassium channels in TE671 human neuroblastoma cells. This intrinsic potassium-channel-blocking (KCB) property is relatively weak (IC50 20 microM), but is not shared by other 5-HT3-receptor ligands including zatosetron, MDL 72222, LY 278, 584, zacopride, 1-phenylbiguanide, and ICS 205-930 (tropisetron). Pre-incubation of the target neuroblastoma cells with several 5-HT-receptor ligands including 5-hydroxytryptamine, 8-OH-DPAT, ketanserin, 2-methyl-5-HT, as well as a number of potent 5-HT3 agonists and antagonists and two selective neurotoxins, failed to abolish the KCB action of ondansetron. A preliminary structure-activity relationship analysis indicates that the KCB activity of ondansetron is almost entirely attributable to its structural nucleus, 2,3-dihyro-9-methyl-4(1H)-carbazolone. It is hypothesized that the KCB action of ondansetron is mediated through receptors other than 5-HT3 receptors. The KCB activity of ondansetron may be a significant factor in the in-vivo cognition-enhancing activities of this compound, conceivably due to depolarization of the hippocampal synaptic membranes and a consequent augmentation of neurotransmission.

Effects of n-dodecylguanidine on A-type potassium channels: role of external surface charges in channel gating.

n-Dodecylguanidine (C12-G) is an amphipathic compound with a guanidine moiety, which is positively charged at physiological pH, and a hydrophobic side chain. Its effects on an A-type K+ channel clone (rKv1.4) expressed in Xenopus oocytes were examined. C12-G caused a concentration-dependent (1-20 microM) positive shift in the voltage dependences of the following channel properties: activation, inactivation, rate of decay during depolarization, and rate of recovery from inactivation. C12-G was effective when added to the bath solution but was without effect when applied to the cytoplasm of oocytes, indicating an extracellular site of action. The effects of C12-G were antagonized by elevation of the extracellular Mg2+ concentration and by external guanidine ions but were augmented by lowering of the ionic strength of the external solution. C12-G did not affect the instantaneous current-voltage relationship for rKv1.4 or the time constant of decay during strong depolarizations, when the voltage dependence of channel activation approached a plateau. Our observations suggest that C12-G exerts its actions by causing a positive shift in the external surface potential around rKv1.4, without altering the ion permeation process or voltage-independent transition steps. In canine ventricular myocytes, C12-G caused changes in the function of a native A-type K+ channel similar to those seen with rKv1.4. However, C12-G had negligible effects on the voltage dependence of the slow delayed-rectifier K+ channel in the same cell type, suggesting that the actions of C12-G are not nonspecific.

Use of cultured human neuroblastoma cells in rapid discovery of the voltage-gated potassium-channel blockers.

Depolarization of human neuroblastoma cells by high concentrations of extracellular potassium ions, leads to the activation of the voltage-gated potassium channels. The activity of such potassium channels can be effectively and rapidly monitored by tracking the efflux of 86Rb from pre-loaded target cells in response to the depolarizing stimulus. The inclusion of compounds with unknown activity in the assay medium, can result in the identification of novel blockers of the voltage-gated potassium channels. Since this functional assay is performed in 96-well microtitre plates, it represents a rapid and high-volume primary screening method for the detection and identification of the voltage-gated potassium-channel blockers, which may have therapeutic utility in several indications including memory degeneration and cardiac arrhythmias.

Cloning of an apamin binding protein of vascular smooth muscle.

The receptor for the bee venom derived neurotoxin, apamin, is widely believed to be an integral component of the small conductance calcium-activated potassium channel in many excitable cells. By affinity chromatography on immobilized apamin, a 78 kD apamin binding protein of the bovine brain synaptosomes was isolated. Antibodies were elicited against this protein and used to clone a cDNA from a porcine vascular smooth muscle expression library. This gene (Kcal 1.8) codes for a 438 amino protein with four potential transmembrane domains, one putative calcium binding site, a protein kinase C phosphorylation site, and a leucine zipper motif. Kcal 1.8 encoded protein has no significant sequence homologies with any known ion channels or receptors. Kcal 1.8 is likely to encode a protein associated with the small conductance calcium-activated potassium channel in vascular smooth muscle.

Screening for potassium channel modulators by a high through-put 86-rubidium efflux assay in a 96-well microtiter plate.

A rapid and sensitive 86Rb efflux assay to detect chemical compounds capable of modulating the ATP-dependent potassium (KATP) channel is described. This assay, which is performed in a 96-well microtiter plate, utilizes a substrate adherent cell line as the target, requires a small amount of 86Rb as the tracer, and is a suitable system for performing the biochemical and pharmacological characterization of the KATP-channel and its activators. Because this assay is amenable to automation, it presents a useful means for high-volume screening of chemical compounds on a routine basis.

Gene expression in Xenopus oocytes.

1. Gene expression in Xenopus oocytes is now an integral part of many molecular cloning strategies. 2. For some genes, such as those encoding the ion channels, this system has emerged as the only available means to authenticate and examine the biological activities of the cloned DNA. 3. This review discusses some of the current applications of Xenopus oocytes in modern molecular biology.

Mast cell degranulating peptide: a multi-functional neurotoxin.

This review discusses our present knowledge of the structure and activities of the mast cell degranulating peptide (MCDP). This peptide is a basic, 22 amino acid residue component of honey bee venom with striking immunological and pharmacological activities. MCDP is a potent anti-inflammatory agent, but at low concentrations it is a strong mediator of mast cell degranulation and histamine release. MCDP is also an epileptogenic neurotoxin, an avid blocker of the potassium channels and can cause a significant lowering of the blood pressure in rats. Some of the biological activities of MCDP appear to have distinct mechanisms and may represent a good illustration of the structure-function relationship.

A heterogeneous double antibody enzyme-linked immunoassay to measure beta-galactosidase fusion protein.

A rapid and sensitive enzyme-linked immunoassay (ELISA) to quantitate recombinant fusion proteins encoded by cloned cDNA in the bacteriophage lambda gt11 is described. Since the fusion protein is expressed in an equimolar ratio to beta-galactosidase, the assay derives the concentration of the recombinant protein in total bacterial lysates or pure preparations from the measurement of beta-galactosidase with an enzyme-linked immunoassay. This assay is a useful technique to measure the recombinant proteins for subsequent immunological and biochemical characterization.

Specific phosphorylation by calcium-EGTA complex of a 75 kDa human tumor plasma membrane protein (pp75).

1. The major functional role played by phosphorylation of plasma membrane proteins in the biological properties of tumor cells suggests that identification of protein kinases and their substrates will contribute to our understanding of the molecular basis of the malignant process and of the aberrant behavior of tumor cells. 2. The present study has investigated the phosphorylation of surface proteins of human tumor cells. Incubation of plasma membranes isolated from cultured human melanoma cells with [gamma-32P]ATP in the presence of Ca2+ and ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) resulted in specific phosphorylation of serine and threonine residues on a 75kDa protein (pp75). 3. Neither Ca2+ or EGTA alone, nor any other divalent metal ion tested could induce phosphorylation of pp75. 4. The phosphorylation of pp75 was directly dependent upon the presence of non-ionic detergents, and was influenced by length of incubation and concentration ratio of Ca2+ and EGTA. 5. Incubation of isolated plasma membranes with [gamma-32P]ATP in the presence of Ca2+ and EGTA and immunochemical analysis by Western blotting with an anti pp75 xenoantiserum detected the pp75 in human melanoma, neuroblastoma, ovarian carcinoma and lymphoid T cells and fibroblasts but not in B-lymphoid cells, renal carcinoma cells, peripheral blood lymphocytes and splenocytes. 6. These results suggest the presence of a new class of plasma membrane bound protein kinases activated by chelated calcium and differentially expressed in normal and transformed human cells.

An immunochemical analysis of the distribution of a brain-specific polypeptide, PEP-19.

Immunochemical and immunohistochemical techniques were used to map the tissue distribution and cellular localization of a rat brain-specific polypeptide, termed PEP-19. PEP-19 was found to be abundant in the cerebellum and olfactory bulbs but was present at much lower levels in other gross brain regions. It was undetectable in all nonneural tissues examined but was present in the cerebellum of several vertebrates, including rat, mouse, guinea pig, monkey, and human. Immunohistochemical analysis revealed that PEP-19 was localized to the soma, axon, and dendritic processes of rat cerebellar Purkinje cells with no demonstrable immunoreactivity in nonneuronal cell types. Furthermore, mutant mice showing degeneration of Purkinje cells exhibit markedly decreased levels of PEP-19. Because PEP-19 appears during the final stages of maturation of Purkinje cells, it may be utilized as a probe to monitor the development of these neurons in vivo.

Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts.

A rapid and efficient method to purify lambda gt11 DNA is described. This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment. The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography. The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification. Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.

Analysis with monoclonal antibodies of the molecular and cellular heterogeneity of human high molecular weight melanoma associated antigen.

Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.

Distinct functional domains on the recombinant human immune interferon molecule.

Two monoclonal antibodies directed against distinct epitopes of recombinant human immune interferon (rIFN-gamma) were used to investigate the relationship between the molecular organization of IFN-gamma and its various biological activities on cultured human melanoma cells. Both monoclonal antibodies inhibited the increase in the expression of cell surface human lymphocyte antigens Class I and II antigens and the antiproliferative and antiviral actions of rIFN-gamma. On the other hand neither monoclonal antibody affected the binding of rIFN-gamma to melanoma cells and its ability to reduce the expression of a high molecular weight-melanoma associated antigen. These data indicate that the functional domains of IFN-gamma responsible for antiviral activity, increased human lymphocyte antigen expression and antiproliferative effects on human melanoma cells may be distinct from that (those) involved in reduced expression of the high molecular weight-melanoma associated antigen and in IFN-gamma binding to cell receptors.

Differential modulation by recombinant immune interferon of the expression and shedding of HLA antigens and melanoma associated antigens by a melanoma cell line resistant to the antiproliferative activity of immune interferon.

By culture of human melanoma Colo 38 cells in the presence of increasing concentrations of recombinant immune interferon (IFN-gamma), the clone RZ gamma-4G.1 resistant to the antiproliferative action of IFN-gamma (3 X 10(4) units/ml) was isolated. This clone was cultured for 6 weeks in the absence of IFN-gamma and was subsequently treated with increasing concentrations of IFN-gamma. Contrary to the response of its parental cell line, treatment of this clone with IFN-gamma did not significantly alter the rate of protein or DNA synthesis and did not markedly modulate the cell surface expression of HLA Class I antigens, of the high molecular weight melanoma associated antigen, and of a Mr 100,000 melanoma associated antigen. IFN-gamma caused an increase in the cell surface expression and in the shedding of HLA Class II antigens from IFN-gamma resistant cells. Four proteins with molecular weights of 32,000, 38,000, 46,000, and 50,000 were induced by IFN-gamma in the parental melanoma cells but not in the resistant clone. Both cell lines bound equivalent amounts of 125I-IFN-gamma to their surface, indicating that the lack of specific surface receptors was not the cause of insensitivity to IFN-gamma. These results indicate that at the cellular level IFN-gamma modulates the expression and shedding of HLA Class II antigens through different mechanisms to those responsible for the antiproliferative action, modulation of the cell surface expression of melanoma associated antigen and of HLA Class I antigens, and induction of new proteins in cultured melanoma cells. Since the success of therapy of malignant diseases with IFN-gamma depends on the extent of resistance of individual tumor cells, the present study may provide a better understanding of the biology of IFN-gamma insensitive tumor cells and particularly the malignant melanoma.

An enzyme-linked double antibody immunoassay to measure murine immunoglobulins--its application to determine the specific activity of radiolabeled monoclonal antibodies.

A sensitive, simple and reproducible biotin-avidin amplified double antibody immunoassay to quantitate low concentrations of mouse immunoglobulins is described. The assay is a useful technique to measure trace levels of murine monoclonal antibodies in culture supernatants of hybridoma cells metabolically labeled with radioactive isotopes. A combination of radioactive counting and measurement of the absorbance of a peroxidase catalyzed reaction permits accurate determination of the specific radioactivity of labeled monoclonal antibodies.