Pinhas Fuchs

Changes Induced in Cell Membranes Adsorbing Animal Viruses, Bacteriophages, and Colicins

Binding of various ligands (hormones, neurotransmitters, immunological stimuli) to membrane receptors induces the following changes: 1. Receptor redistribution (cluste ring, “capping”) 2. Conformational changes that can be detected by fluorescent probes 3. Alteration in membrane fluidity (spin label and fluorescence polarization probes) 4. Changes in fluxes of ions and metabolites 5. Increased phospholipid turnover (especially of phosphatidyl inositol) 6. Activation ofmembrane-bound enzymes (adenyl cyclase, ATPase, transmethylases).

Initial Effects of Viral Infection in Bacterial and Animal Host Cells

Publisher Summary Living organisms are composed of cell societies. The orderly function of these societies, required for the growth and maintenance of the organisms, is regulated by a variety of signals. These signals operate at the level of whole organism (nervous system, blood system, and hormones), at the level of a tissue (embryonal inducers, chalones) , or at the level of the cell (regulation of metabolic functions and cell division). A different type of language and instructions is encountered in the interactions between viruses and the cells infected by them. One can differentiate between two types of signals: first type, well known and extensively studied, involves the instructions inserted by the infecting virus into the infected host cell-instructions in the form of viral nucleic acid, which are then translated by the host cell synthetic machinery into thousands of complete and functional copies of the infecting virus. The other type of signal seems to operate at the surface of the host at the time of adsorption of the virion (or its protein subunits) to the specific receptor on the cell surface. This type of signal may have a formal similarity to the hormone-CAMP interaction, the antigen-antibody interaction, and the lectins-cell membrane interactions. There is no question that membranes, both external and internal, fulfill a very important role in the process of infection by viruses. Most considerations presented in this chapter are concerned with the elucidation of this second type of virus-cell interaction.

Effects of phencyclidine and analog drugs on acetylcholine receptor of cultured muscle cells

Myotubes grown in culture provided a convenient experimental system for the study of the effects of phencyclidine (PCP) and analog drugs on both acetylcholine receptor (AChR) function and on its binding properties. The extent of PCP retention by these cells was studied on the same preparations. PCP, N-ethyl-l-phenylcyclohexylamine (PCE), PCP methiodide (PCPMeI), 1-[1-(3-aminophenyl)-cyclohexyl] piperidine (NH2PCP) and 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) were found to inhibit carbamylcholine (CbCh)-induced 22Na and 45Ca ion fluxes with 50% inhibition (I50) at 2-6 microM drug concentration. The I50 for CbCh-induced 42K+ efflux was 8-20 microM. Ketamine was less efficient with an I50 of 100 microM. Binding of [125I] alpha-bungarotoxin [( 125I]alpha-BGT) was not affected at drug concentrations that cause 100% inhibition of ion fluxes. Retention of [3H]PCP by the myotubes was a saturable process with half-maximal saturation at approximately 20 microM PCP. It was inhibited by PCP and several tertiary analogs, with and I50 of approximately 20 microM. PCPMeI was much less effective, with an I50 of 1 mM. PCPMeI was, however, as potent as PCP in its inhibition of the AChR function although the amount retained by the cells was 50-fold lower than that of PCP. These results are consistent with the theory that PCP and analog drugs affect AChR at a site other than the alpha-BGT binding site, possibly at the ionic channel of the nicotinic receptor.

Interaction of phencyclidine with mouse neuroblastoma cells

Growth of mouse neuroblastoma (Nb) cell (clone M1) was not affected by phencyclidine (PCP) concentrations of 10(-6)M up to 2 x 10(-4)M, whereas 10(-3)M PCP caused a 100% inhibition of cell growth. Several PCP analogs, including the quaternary PCP methiodide, exerted effects similar to those of PCP. The uptake of [piperidyl-3,4-3H]PCP ([3H]PCP) by the Nb cells was studied using cell monolayers in Petri dishes. Non-specific entry of PCP into the cells was linear with added substrate but specific uptake exhibited saturation kinetics. The concentration for half-maximum specific uptake was 2 x 10-(5)M, and the capacity of the cells at saturation was 2-3 nmoles [3H]PCP/mg protein, at 22 degrees. The uptake rate constant was 0.2 +/- 0.05 x 10(5) (M-1 min-1) and the dissociation constant was 0.25 +/- 0.05 (min-1). Uptake was temperature dependent and was inhibited by 2,4-dinitrophenol (DNP). This may indicate that this binding represents (at least in part) an active uptake process of PCP into the cells.

Interaction of Phencyclidines with Acetylcholine Receptor in Cultured Myotubes

Several laboratories have shown that phencyclidine (PCP) and analog drugs interact with the ionic channel of the nicotinic acetylcholine receptor (AChR). These studies were done on two separate preparations, animal organs for electrophysiological studies and membrane preparations for biochemical studies. This work, however, was performed with myotubes grown in culture: these provide a convenient experimental system for the study of PCP and analog drug effects on both receptor function and receptor binding properties. Moreover, the extent of PCP retention by these cells was studied on the same preparations. All experiments were performed on viable cells in petri dishes. PCP and four PCP analogs were found to inhibit carbamylcholine induced 22Na and 45Ca ion fluxes with 50% inhibition (I50) at 2–6 μM drug concentration. The I50 for carbamylcholine induced 42K efflux was 8–20 μM. Ketamine was less efficient with an I50 of 100 μM. 125 I-α-Bungarotoxin binding was not affected at drug concentrations that cause 100% inhibition of ion fluxes. Uptake of 3H-PCP by the myotubes was a saturable process with half maximal saturation at ~ 20 μM PCP. It was inhibited by PCP and by several tertiary PCP analogs, with an I50 of ~ 20 μM. The quaternary analog PCP-methiodide (PCPMeI) however, was much less effective with an I50 of 1 mM. The capacity for 3H-PCP retention by the cells was 4 nmol/mg protein. PCPMeI was as potent as PCP in its inhibition of the AChR function although the amount retained by the cells was 50-fold lower than that of PCP. These results are consistent with the theory that PCP and analog drugs affect AChR function at a site other than the α-Bungarotoxin binding site, possibly at the ionic channel of the nicotinic receptor.