Real-time detection of mitochondrial inhibition at frog motor nerve terminals using increases in the spatial variance in probability of transmitter release.

The effects of Hg2+, methyl mercury, and flufenamic acid, all of which inhibit mitochondria, were examined at frog motor nerve terminals. Unbiased estimates of m (no. of transmitter quanta released), n (no. of functional release sites), p (probability of release), and vars p (spatial variance in p) were obtained using K(+)-induced asynchronous neurosecretion (m, n and p not having the same definitions as with nerve-evoked release). Transient but significant increases in m, n, p and vars p were found with all three agents. These findings indicate that mitochondrial inhibition and release of sequestered Ca2+ can be detected as a real-time increase in vars p. The results also suggest that changes in vars p might be used to differentiate between cellular (membrane) and subcellular (organellar) actions of drugs at the nerve terminal.

Effect of the putative cognitive enhancer, linopirdine (DuP 996), on quantal parameters of acetylcholine release at the frog neuromuscular junction.

1. The subcellular mechanism and site of action of linopirdine or DuP 996 (3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) was investigated at the frog neuromuscular junction, using miniature endplate potential (m.e.p.p.) counts and a new method for obtaining unbiased estimates of n (number of functional release sites), p (probability of release), and varsp (spatial variance in p). 2. DuP 996 produced an increase in m (no. of quanta released), which was due to an increase in n and p. The increase in m was concentration-dependent over a range of 0.1-100 microM and completely reversible with 15 min of wash. There was a saturation in the increase in p, but not in the increase in m and n, for [DuP 996] > 10 microM. By contrast, there was no major change in varsp. 3. Block of presynaptic Na(+)- and Ca(2+)-channels with 3 microM tetrodoxin and 1.8 mM Co2+ prevented the m.e.p.p. frequency increase to DuP996, and this effect was completely reversed by washing. 4. Application of the neuronal Ca(2+)-channel blocker, omega-conotoxin GVIA (1 microM) brought about a rapid and profound decrease in the m.e.p.p. frequency increased produced by DuP996. The effect of the toxin was not reversed by prolonged washing. 5. Block of voltage-gated K(+)-channels with 100 microM 4-aminopyridine (4-AP) resulted in only a small (28%) increase in m. The combination of 4-AP (100 microM) and DuP996 (10 microM) produced an increase in m (189%) which was much greater than the sum of the responses to each agent alone. This increase in m was due solely to an increase in n, as p and varsp were unchanged.6. For [DuP 996] up to 100 gM, there was no apparent change in the mean size, amplitude distribution,or time course of m.e.p.ps, signifying that it had no anticholinesterase activity.7. It is concluded that DuP 996 increases the release of quantal transmitter but not the postsynaptic response to the quanta. This appears to involve an effect at the nerve terminal membrane, most likely an increase in Ca2+-conductance, and not an action to block K+-conductance or to release Ca2+ from intraterminal organelles.

Unbiased estimates of quantal release parameters and spatial variation in the probability of neurosecretion.

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n) and probability of release (p), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 microM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La(3+)-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n and p and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.

Aluminum inhibits glutamate release from transverse rat hippocampal slices: role of G proteins, Ca channels and protein kinase C.

Aluminum (Al) has been shown to produce deficits in learning and memory. The present experiments tested the hypothesis that Al-induced inhibition of learning may be due to its effect on glutamate release secondary to changes in calcium channel function and/or intracellular events triggering glutamate release. Calcium-dependent potassium (K)-evoked [14C]-glutamate release from 400 microns transverse rat hippocampal slices was inhibited by Al in a concentration dependent manner (IC50 = 40 microM). Aluminum (30, 100 microM) noncompetitively inhibited Bay K 8644-evoked glutamate release. 4-Aminopyridine (30, 1000 microM) noncompetitively attenuated the Al inhibition of glutamate release, suggesting an Al-induced alteration of Ca channel function. Activation of the Gi protein by R(-)phenylisopropyladenosine (PIA; 1 microM) reduced K-evoked glutamate release 69%, whereas 300 microM Al produced an 84% reduction. These effects were prevented by the Gi protein inhibitor N-ethylmaleimide (NEM; 100 microM), suggesting an effect of Al on the Gi protein to inhibit glutamate release. Phorbol myristate acetate (0.16 microM)-induced glutamate release was inhibited by 300 microM Al and 80 microM polymyxin B, suggesting an Al modulation of protein kinase C (PKC)-evoked glutamate release. These results demonstrate an Al inhibition of glutamate release that may be mediated by multiple, but interconnected mechanisms (e.g., via interactions with Ca systems), providing multiple targets for an Al-induced alteration of neuronal function.

Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.

Tetrahydroaminoacridine and physostigmine have opposing effects on probability of transmitter release at the frog neuromuscular junction.

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 microM) caused an increase in m (no. of quanta released) as measured by K(+)-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 microM. By contrast, physostigmine (3 microM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

Reduced intestinal calcium and dietary calcium intake, increased aluminum absorption, and tissue concentration in the rat.

To test the influence of calcium (Ca) on aluminum (Al) absorption, Ca was withheld from or added (1mM) to the perfusate of the in situ rat gut. The rats had been maintained on Purina Rat Chow. Ca addition significantly decreased (to 70%) the rate of Al disappearance from the gut and decreased (to 55%) the area under the curve of Al appearance in portal blood. To test the influence of Ca deficiency on Al absorption, rats were maintained on a low-Ca (0.008%) or a Ca-replete (0.5%) diet for 1-4 wk. The in situ gut was prepared, and a perfusate containing approximately 1 microM Ca was used. The rate of Al disappearance from the gut of low-Ca diet rats was significantly faster than from the gut of rats maintained on the Ca-replete diet, averaging 156% of the latter. Al appearance in portal blood was significantly greater (averaging 38%) in rats maintained on the low-Ca diet than in controls. To determine if Ca deficiency influences Al tissue distribution independent of gastrointestinal Al absorption, rats maintained on a low-Ca or a Ca-replete diet received 20 ip Al injections over 1 mo. Rats eating the low-Ca diet demonstrated enhanced tissue Al accumulation in all tissues studied, except for muscle and cerebral cortex. These results demonstrate enhanced Al absorption and tissue retention in the presence of reduced intestinal Ca concentration and reduced Ca intake.

Aluminum uptake by the in situ rat gut preparation.

An in situ rat gut preparation was used to elucidate the mechanisms of gastrointestinal aluminum (Al) absorption. Al uptake rate at the mucosal surface was decreased by the paracellular pathway blockers kinetin (1 mM) and 2,4,6-triaminopyrimidinium (10 mM), by sodium removal with choline substitution and by treatment with amiloride (1 mM), an epithelial sodium transport blocker. The rate of Al uptake was unchanged by 2,4-dinitrophenol (0.1 mM), 4-aminopyridine (0.1 mM, 0.5 mM) and verapamil (0.1 mM). The rate of Al uptake was increased from a medium containing no added calcium, a treatment which decreases resistance to flux in the paracellular pathway. These results suggest that gastrointestinal Al uptake occurs by an energy-independent, sodium-dependent, paracellular pathway-mediated process.

Influence of calcium on aluminum accumulation by the rat jejunal slice.

The isolated rat jejunal slice was used to determine if aluminum (Al) interacts with the gastrointestinal (GI) calcium (Ca) transporting system. Al uptake by the rat jejunal slice was reduced by Ca channel blockers (verapamil, nifedipine, diltiazem-10 microM) and a medium containing no added Ca. Conversely, Al uptake was increased by Ca channel activators (4-aminopyridine, .05mM, .1mM; Bay k 8644, 1, 10 microM) and by 5mM Ca. Al uptake was saturable and energy dependent but yielded a low activation energy (Ea = 3.9 +/- 0.3 kcal/mole). Al uptake was increased by vanadate (100 microM), an inhibitor of both the active Ca pump and Na/K-ATPase. These results suggest that Al does interact with the GI Ca transporting system. This interaction may form the basis for its accumulation and toxicity in different tissues which contain similar processes for handling Ca.

Aluminum intoxication and the victim of Alzheimer's disease: similarities and differences.

Aluminum (Al) has been implicated in the pathogenesis and may produce a model of senile dementia of the Alzheimer's type (SDAT). To better understand the effects of Al on the mammalian brain, Al was studied in rabbits and rats using several experimental approaches. Similar behavioral toxicity and site and degree of neurofibrillary tangle (NFT) development were obtained after intracerebroventricular (icv) or repeated sc Al, although a more protracted time course after the latter. SDAT victims demonstrate some similar behavioral signs but a different type of NFT. A classically conditioned, defensive reflex (nictitating membrane extension) was used to compare response acquisition, retention, and extinction in Al-exposed and control rabbits of various ages. Both increasing age and Al exposure attenuated these measures, suggesting that advanced age and Al intoxication provide behavioral models of SDAT. A deficit in acquisition of a classically conditioned response (eyeblink) in senile-demented patients has been reported. These cognitive deficits in rabbits occurred after a less than two-fold increase in brain Al. Brain Al in SDAT victims has been reported to be unchanged or slightly increased. Further similarity to SDAT was obtained with 4-aminopyridine (4-AP) which attenuated Al-induced behavioral deficits in rabbits and the Al inhibition of glutamate release from rat hippocampal slices. 4-Aminopyridine has been reported to attenuate behavioral deficits in SDAT patients. A tubulin, GTP binding site was blocked in SDAT victim brain but not in control or Al-intoxicated rabbit brain, indicating a biochemical dissimilarity. In summary, Al-intoxication produces a model of SDAT with many similarities but significant differences.