Potassium-stimulated 45Ca entry into rat brain synaptosomes was measured at times ranging from 1 to 60 s. The K-rich solutions were used to depolarize the synaptosomes. Backflux of 45Ca from the synaptosomes was negligible during the first 10-20 s of incubation. An initial ("fast") phase of K-stimulated Ca entry, lasting from 1 to 2 s was observed. This phase was inhibited by low concentrations of La (KI approximately equal to 0.3 microM). It was also abolished ("inactivated") by incubating the synaptosomes in depolarizing solutions (containing veratridine, gramicidin, or elevated [K]o) before the addition of 45Ca. An additional long lasting ("slow") phase of K-stimulated Ca entry was also detected. This "slow" Ca entry was much less sensitive to La (KI > 100 microM) and was not affected by depolarizing the synaptosomes before the addition of 45Ca. The rate of influx during the fast phase was about four times the rate of Ca influx during the slow phase. Neither the fast nor slow phase of Ca entry was sensitive to tetrodotoxin (10 microM), a potent blocker of Na channels, but both phases were inhibited by Ni, Mn, Mg, and other agents that block Ca channels. The data are consistent with the presence of two distinct populations of voltage-regulated, divalent cation-selective pathways for Ca entry in presynaptic brain nerve endings.

SUMMARY1. The uptake and efflux of 54Mn and 45Ca, and the release of dopamine (DA) were measured in pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain.2. The uptake of Mn and Ca was increased when forebrain or striatal synaptosomes were incubated in a depolarizing, K-rich solution. The time courses of K-stimulated Mn and Ca entry were similar: there was initially a high rate of ion accumulation, lasting 1-3 s, that gradually levelled off. The initial uptake of Mn, like that of Ca, was greatly diminished by a 10 s pre-incubation in K-rich solution prior to the addition of radiotracer.3. Several Ca channel blockers, including Ni (0 03 mM), Sr (2'0 mM), Co (0 04 mM), Ba (1-5 mM) and La (0-2 mM), suppressed the K-stimulated uptake of Mn and of Ca to a similar extent. The K-stimulated uptake of Mn increased as a function of the external Mn concentration, and saturated at high external concentrations of Mn. These high concentrations of Mn also blocked the K-stimulated uptake of Ca.4. There was a decreased efflux of Ca, but not of Mn, from the synaptosomes when the external Na concentration was reduced. The Na-dependent efflux of Ca was diminished by external Mn, but was unaffected when the synaptosomes were loaded with Mn.5. The rate of [3H]DA release from striatal synaptosomes was less than 0-001 sin non-depolarizing, low-K solutions, in the absence or presence of Mn and Ca (1 mM). The rate ofrelease was also unchanged in depolarizing, K-rich solutions in the absence of these divalent cations. The addition of 1 mM-Mn to a K-rich solution increased the rate of DA release by about 40 %, and the time course of release was linear for at least 30 s. The addition of t mM-Ca increased the rate of release nearly 100-fold during the first second, and thereafter the rate of release rapidly declined.6. Ni (1 mM) and, to a lesser extent, Mg (10 mM) reduced the rate of K-stimulated DA release that is dependent on either Mn or Ca. The pattern of inhibition of DA release resembled the pattern of inhibition of K-stimulated uptake of Mn and Ca.

SUMMARY1. The regulation of cytosolic Ca concentration ([Ca]i) was studied with the fluorescent Ca indicator, quin2, in pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain. [Ca]i increases by about 2-fold when the external Na concentration is lowered from 145 to 5 mm, and returns to its initial level when external Na is restored. This recovery occurs also in the presence of FCCP.5. These results suggest that Na/Ca exchange, but not mitochondrial Ca uptake, plays a role in regulating [Ca]i and in allowing the nerve terminals to recover from Ca loading.

We examined the effects of extracellular and intracellular pH changes on the influx of radioactive 4~Ca, the concentration of ionized Ca (pCa0 as monitored with the Ca-sensitive fluorescent indicator fura-2, and the efflux of dopamine in presynaptic nerve endings (synaptosomes) isolated from rat brain corpora striata and preloaded with [3H]dopamine. Cytosolic pH (pHi) was monitored by loading the synaptosomes with the H+-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) (see Nachshen, D. A., and P. Drapeau, 1988, Journal of General Physiology, 91:289-303). An abrupt decrease of the pH of the external medium, from 7.4 to 5.5, produced a slow decrease of pHi (over a 5-min period) from an initial value of 7.2 to a steady state level of-5.8. When 20 mM acetate was present in acidic media, pH~ dropped as fast as could be measured (within 2 s) to a level similar to that reached (more slowly) in the absence of acetate. It was therefore possible to lower pH~ over short time periods to different levels depending on whether or not acetate was present upon extracellular acidification. Extracellular acidification to pH 5.5 (in the absence of acetate) had no significant effect on pCai and dopamine release over a 30-s period (pHi = 6.4). Acidification in the presence of acetate lowered pHi to 5.8 without affecting pCa~, but dopamine efflux increased -20-fold. This increase in basal dopamine release was also observed in the absence of extracellular Ca. Thus, intraterminal, but not extracellular, acidification could stimulate the efflux of dopamine in a Caindependent manner. The high Q~0 (5.6) of acid-stimulated dopamine efflux in the presence of nomifensine (which blocks the dopamine carrier) was consistent with an activation of vesicular dopamine release by H § When synaptosomes were both depolarized for 2 s in high-K (77.5 mM) solutions and acidified (in the absence of acetate), there was a parallel block of 45Ca entry and evoked dopamine release (50% block at pH 6.0 with 0.2 mM external Ca). When

K-stimulated (voltage-dependent) influx of "Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain . Influx was terminated at I s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D . A ., and Blaustein, M . P ., 1980, J. Gen. Physiol ., 76 :709-728) . This influx was blocked by multivalent cations with half-inhibition constants (K I ) that clustered in three distinct groups : (a) K, > I mM (Mg", Sr", and Bat+ ) ; (b) K, = 30-100 AM (Mn 2+, Co t+ , Ni t+ , Cu e+ , Zn2+ , and H g 2+) ; ( c ) K, < I AM (Cd2+, Y s+, Las+ and the trivalent lanthanides, and Ph 2+) . Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization . The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s ; inhibition was similar after 1 s or 30 min of exposure to these ions . The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation . The relative efficacies of the lanthanides as fast channel blockers were compared ; there was a decrease in inhibitory potency with decreasing ionic radius . A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations . The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them . Small cations are unable to bind effectively to both anions . The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel .Address reprint requests to Dr .