Zvi Vogel

Differential Modulation of Adenylyl Cyclases I and II by Various Gβ Subunits

The accepted dogma concerning the regulation of adenylyl cyclase (AC) activity by Gβγ dimers states that the various isoforms of AC respond differently to the presence of free Gβγ. It has been demonstrated that AC I activity is inhibited and AC II activity is stimulated by Gβγ subunits. This result does not address the possible differences in modulation that may exist among the different Gβγ heterodimers. Six isoforms of Gβ and 12 isoforms of Gγ have been cloned to date. We have established a cell transfection system in which Gβ and Gγ cDNAs were cotransfected with either AC isoform I or II and the activity of these isoforms was determined. We found that while AC I activity was inhibited by both Gβ1/γ2 and Gβ5/γ2 combinations, AC II responded differentially and was stimulated by Gβ1/γ2 and inhibited by Gβ5/γ2. This finding demonstrates differential modulatory activity by different combinations of Gβγ on the same AC isoform and demonstrates another level of complexity within the AC signaling system.

'Aplysia' Acetylcholine Receptors: Blockade by and Binding of alpha- Bungarotoxin

Summary αBT is a snake toxin which in vertebrate systems binds with great specificity to ACh receptors. We have studied the effects of αBT on the electrophysiological response to ACh of identifiable cells in the nervous system of the marine mollusc, Aplysia, and the binding of [125I]αBT to a ganglionic preparation. Aplysia has 3 pharmacologically distinct ACh responses and each causes a different conductance change. αBT blocks all 3 responses of iontophoretically applied ACh. In all cases the inhibition is reversed on washing. Binding of [125I]αBT to the ganglionic preparation is a saturable process. The dissociation constant of binding calculated from rates of association and dissociation of the toxin-receptor complex is0.8 × 10−9 M. Binding of [125I]αBT is inhibited by unlabeled toxin, ACh agonists, and antagonists as well as by eserine, ouabain, and TEA but not by the transmitters serotonin and dopamine.

Localization of α-bungarotoxin binding sites in synapses of the developing chick retina

Abstract Alpha-bungarotoxin binding sites in chick retina from 12 days in ovo to hatching, were visualized at the ultrastructural level by use of a 1:1 conjugate of horseradish peroxidase with α-bungarotoxin. At all stages binding sites in the inner plexiform layer were localized in synapses, predominantly on or near the postsynaptic membrane. Localization of binding sites was found in bipolar and amacrine-cell synapses which appeared morphologically immature as well as more well developed synapses. The results suggest that α-bungarotoxin-binding synapses, tentatively considered to be nicotinic cholinergic, are formed throughout the course of synaptogenesis, and that the aggregation of nicotinic receptors occurs early in the formation of these synapses.

Association of laminin and other basement membrane components with regions of high acetylcholine receptor density on cultured myotubes.

The distribution of immunoreactivity to basement membrane components in cultures of rat skeletal myotubes was compared to acetylcholine receptor distribution by fluorescence microscopy. Laminin occurred in patches on the myotube surface, and most laminin patches coincided or overlapped with acetylcholine receptor aggregates. Almost all receptor aggregates coincided with laminin patches. Most of the heparan sulfate proteoglycan and fibronectin was associated with non‐muscle cells, but some patches coincided with receptor aggregates on myotubes. In cultures treated with l‐ascorbate, collagen types IV and V covered much of the myotube surface and receptor aggregates often coincided with intense collagen patches. When receptor aggregation was induced by treatment of cultures with soluble neural factors, the newly formed receptor aggregates coincided with laminin patches.

IMMUNOPEROXIDASE LOCALIZATION OF ALPHA BUNGAROTOXIN: A NEW APPROACH TO MYASTHENIA GRAVIS*

The IPBT method has made it possible to precisely visualize the AChR. Normal distribution of AChR is at the peaks of the postjunctional folds of the muscle sarcolemmal membrane with a small amount present on the axonal tip as well. Denervated muscle fibers have extrajunctional AChR. In MG, there are also denervated-appearing fibers but these do not have extrajuctional AChR with the IPBT stain. To explain this, we have been able to demonstrate a serum factor capable of blocking the binding of alpha-BuTx to the AChR and have shown for the first time that this factor is capable of acting at the neuromuscular junction itself. This blocking factor may play a major role in causing the weakness of MG.