PubMed · 4950075
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T J Biscoe. 1971. Carotid body: structure and function.. https://doi.org/10.1152/physrev.1971.51.3.437
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1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.
1. The contribution of gap junctions to endothelium-dependent relaxation was investigated in isolated rabbit conduit artery preparations pre-constricted by 10 microM phenylephrine (PhE). 2. Acetylcholine (ACh) relaxed the thoracic aorta by approximately 60 % and the superior mesenteric artery (SMA) by approximately 90 %. A peptide possessing sequence homology with extracellular loop 2 of connexin 43 (Gap 27, 300 microM) inhibited relaxation by approximately 40 % in both artery types. Gap 27 also attenuated the endothelium-dependent component of the relaxation induced by ATP in thoracic aorta but did not modify force development in response to PhE. 3. NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of NO synthase, attenuated ACh-induced relaxation by approximately 90 % in the aorta but only by approximately 40 % in SMA (P < 0.05). Residual L-NAME-insensitive relaxations were almost abolished by 300 microM Gap 27 in aorta and inhibited in a concentration-dependent fashion in SMA (approximately 50 % at 100 microM and approximately 80 % at 10 mM). Gap 27 similarly attenuated the endothelium-dependent component of L-NAME-insensitive relaxations to ATP in aorta. 4. Responses to cyclopiazonic acid, which stimulates endothelium-dependent relaxation through a receptor-independent mechanism, were also attenuated by Gap 27, whereas this peptide exerted no effect on the NO-mediated relaxation induced by sodium nitroprusside in preparations denuded of endothelium. 5. ACh-induced relaxation of 'sandwich' mounts of aorta or SMA were unaffected by Gap 27 but completely abolished by L-NAME. 6. We conclude that direct heterocellular communication between the endothelium and smooth muscle contributes to endothelium-dependent relaxations evoked by both receptor-dependent and -independent mechanisms. The inhibitory effects of Gap 27 peptide do not involve homocellular communication within the vessel wall or modulation of NO release or action.
The human homologue of the vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the vesicular monoamine transporter (hVMAT2) are differentially targeted to two populations of regulated secretory organelles when expressed in PC12 cells. Western blot analysis of subcellular fractions from sucrose equilibrium density gradients and glycerol velocity gradients of homogenates from stably transfected cells revealed hVAChT immunoreactivity in fractions that contain synaptophysin, a marker of synaptic vesicles, while hVMAT2 immunoreactivity was confined to heavy fractions containing chromogranin B, a marker of large dense core vesicles. In cells treated with nerve growth factor, hVAChT immunoreactivity alone co-localized with synaptophysin and was abundantly expressed on synaptic vesicle clusters. Chimeras between hVMAT2 and hVAChT were utilized to identify the domain of hVAChT required for its expression on synaptic vesicles and which would shift the expression of hVMAT2 from large dense core vesicles to synaptic vesicles. Biochemical, immunocytochemical, and electron microscopic analyses revealed that a chimera in which the cytoplasmic tail of hVMAT2 was replaced with hVAChT sequences was now preferentially targeted to synaptic vesicles. In addition, hVAChT expression on synaptic vesicles was nearly abolished when the hVMAT2 cytoplasmic tail was present. Thus, structural information resides within the terminal cytoplasmic domain of VAChT, which specifically targets it to synaptic vesicles.