J. B. Suszkiw

SELECTIVE LOCALIZATION OF A HIGH AFFINITY CHOLINE UPTAKE SYSTEM AND ITS ROLE IN ACh FORMATTON IN CHOLINERGIC NERVE TERMINALS

The avian iris‐ciliary nerve preparation exhibits two distinct choline uptake systems. One component, a sodium dependent, high affinity system Km‐2 am and Vmax ‐ 0.5 pmolpin per preparation is confined to nerve terminals. The other component is localized in muscle cells. It is sodium independent and low affinity system (Km ‐ 200 am and Vmax ‐ 16 pmol/min per muscle). The high affinity uptake of choline and the synthesis of ACh in the nerve terminals are coupled. Vmax Ach formation ‐0.5 pmol/min. is the same as Vmax for choline transport; however. with the external choline concentration equal to that of avian plasma only ‐50% of choline taken up is converted to ACh. In contrast to the nerve terminals, the cell bodies of the same neurons are deficient in the high affinity uptake‐ACh synthesis coupled system. This indicates a nerve terminal membrane specialization related to neuro‐transmitter synthesis.

Further Characterization of Phasic Calcium Influx in Rat Cerebrocortical Synaptosomes: Inferences Regarding Calcium Channel Type(s) in Nerve Endings

Abstract Under conditions minimizing the contribution of Na+/Ca2+ exchange to calcium entry in synaptosomes, the K+ depolarization‐dependent calcium influx (JCa) is a single exponential function of time. JCa activates and slowly inactivates at membrane potentials positive to –50 mV, a result indicating the involvement of moderate voltage‐activating, slowly inactivating calcium channels. Calcium channels in synaptosomes are characterized by stronger sensitivity to blockage by Cd2+ than Co2+, insensitivity to dihydropyridine calcium antagonists or the agonist Bay K 8644, and weak, partial sensitivity to the peptide toxin ω‐conotoxin GVIA. These characteristics suggest that voltage‐sensitive calcium channels in rat cerebrocortical synaptosomes are dissimilar from the somatic T, N, or L channel types. JCa is not affected by treatment of synaptosomes with the adenylate cyclase activator forskolin, the membrane permeant dibutyryl‐cyclic AMP, or the kinase C activator phorbol 12‐myristate 13‐acetate diester, results suggesting that calcium channels in synaptosomes are not directly modulated by protein kinase A‐ or C‐mediated phosphorylation.

VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [3H]acetate as a precursor of ACh. Little vesicular [3H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post‐stimulation recovery the newly synthesized [3H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [3H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.

Permeation of Pb2+ through calcium channels: fura-2 measurements of voltage- and dihydropyridine-sensitive Pb2+ entry in isolated bovine chromaffin cells

Fura-2 was used to monitor Pb2+ entry into isolated bovine chromaffin cells exposed to micromolar concentrations of Pb2+ in media containing basal or high concentrations of K+. The entry of Pb2+ consists of voltage-independent and voltage-dependent (K(+)-stimulated) components. The voltage-dependent Pb2+ entry is enhanced by Ca2+ channel agonist BAY K 8644 and blocked by the channel antagonist nifedipine, suggesting the involvement of the L-type Ca2+ channels. In contrast to the transient, K(+)-depolarization-dependent increase in [Ca2+]i, the increase in [Pb2+]i is sustained over a period of several minutes, suggesting the absence of channel inactivation and/or the saturation of Pb(2+)-buffering capacity of the cell cytosol.

Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

Abstract: We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb2+‐induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high‐affinity (KA(Pb) = 2.4 × 10−12M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar‐(Ki = 7.1 × 10−9M) and micromolar‐ (K′i = 2.8 × 10−7M) affinity sites, respectively. Activation of PKC by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Ca2+‐deficient, Pb2+‐containing medium, enhances the Pb2+‐induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half‐maximal activation of the secretory response from 7.5 × 10−10 to 5.7 × 10−11M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb2+‐induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb2+‐triggered secretion.

Heterogeneity of presynaptic calcium channels revealed by species differences in the sensitivity of synaptosomal 45Ca entry to ω-conotoxin

The inhibition of K+-depolarization dependent Ca influx by omega-conotoxin GVIA was compared in the frog, chick, and rat brain synaptosomes. The toxin at concentrations greater than or equal to 0.3 microM completely inhibited Ca entry in the frog and chick preparations, but was only partly effective in blocking Ca influx in the rat brain synaptosomes. In chick synaptosomes the toxins effect was biphasic: a small component (approximately equal to 15%) of total Ca influx was inhibited by the toxin with high affinity (I50 less than 0.002 microM); a major component (approximately equal to 80%) of Ca influx was inhibited with a moderate affinity (I50 approximately equal to 0.05 microM). In rat brain synaptosomes 40% of Ca influx was inhibited by the toxin with low affinity (I50 approximately equal to 0.3 microM), and 60% of Ca influx was unaffected by the toxin concentration of up to 10 microM. These data suggest a heterogeneity of voltage-sensitive Ca channels in vertebrate brain synaptosomes.

CHOLINE UPTAKE BY CHOLINERGIC NEURON CELL SOMAS

The cellular compartments of ciliary ganglia take up choline by a single, saturable process with Km=7.1 × 10−5 M and Vmax= 4.66 pmol/min per ganglion: Denervation of the ganglia and the resultant degeneration of nerve terminals caused no significant decrease of the rate of accumulation of choline by the ganglia. This indicates that the measured uptake is by the postganglionic ncurons and nonneural elements (NNE: glial and connective tissue cells) in the ganglia. This uptakc is not dependent on metabolic energy and is not affectcd by lowcring Na+ or raising K+ concentrations in the incubating mcdia but is depressed in the presence of ouabain and hemicholinium‐3. The presence or Na+‐dependent. rapidly saturable uptake in the preganglionic nerve terminals which is not detectablc kinetically is, however, inferred from a decrease in ACh synthesis in dcncrvatcd prcparations and a similar decrcasc in intact ganglia incubated in low Na+ solution.

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

Abstract: The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin‐permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 μM Pb2+ to intact synaptosomes in Ca2+‐deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2+‐induced ACh release can be augmented by activation of the voltage‐gated Ca2+ channels in nerve terminals. The lead‐induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1–50 nM (EC50± 4 nM) free Pb2+ to digitonin‐permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2–5 μM (EC50± 0.5 μM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.

Temporal characteristics of potassium-stimulated acetylcholine release and inactivation of calcium influx in rat brain synaptosomes.

Abstract: The time course of Ca2+‐dependent [3H]acetylcholine ([3H]ACh) release and inactivation of 45Ca2+ entry were examined in rat brain synaptosomes depolarized by 45 mM [K+]o. Under conditions where the intrasynaptosomal stores of releasable [3H]ACh were neither exhausted nor replenished in the course of stimulation, the K+‐evoked release consisted of a major (40% of the releasable [3H]ACh pool), rapidly terminating phase (t1/2= 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K+‐stimulated Ca2+ entry suggests the presence of fast‐inactivating, slow‐inactivating, and noninactivating, or very slowly inactivating, components. The fast‐inactivating component of the K+‐stimulated Ca2+ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K+ stimulus. The noninactivating Ca2+ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K+]o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage‐dependent Ca channels.

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure.

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30-40% reduction of [3H]hemicholinium-3 ([3H]HC-3) binding in the hippocampus through PN120, and 20-30% reduction of septal and hippocampal choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [3H]quinuclidinyl benzylate ([3H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56-PN112. Neither short- nor long-term effects of Pb exposure on [3H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50-90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampal TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.