Regulation Of Calcium Entry Research Articles

Intracellular calcium gradients in cultured human uterine smooth muscle: a functionally important subplasmalemmal space

The plasma membrane contains the key elements for the control of coupling excitation to contraction in smooth muscle. The superficial calcium buffer barrier, initially proposed by van Breemen for vascular smooth muscle, may participate in the regulation of calcium entry in other smooth muscle types. To investigate the relationship between the sarcoplasmic reticulum (SR) and the plasma membrane in myometrial smooth muscle cells, we performed experiments using videofluorescence imaging and cell-attached electrophysiology. The cell-attached patch was used as a reporter for the free calcium in the subplasmalemmal space by monitoring openings of the Maxi-K channel. Calcium green-1 was used to simultaneously monitor changes of the deep cytosolic calcium con centrations. The cell with the patch attached was stimulated via an intercellular calcium wave from an adjacent cell. In this fashion, release of SR calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space of the cell studied. With physiological bathing solution, six of seven calcium waves activated Maxi-K channels. Surprisingly, the Maxi-K channels began opening 6.3 ± 4.7s (range 2.6–15.0s) after the wave passed the pipette location. When plasma membrane calcium fluxes were inhibited with 100μM lanthanum, no Maxi-K channel openings were observed in six of seven experiments. These results are best explained by a subplasmalemmal space in which the calcium concentration is largely controlled by store-operated channels. These results suggest the superficial buffer barrier as merely one aspect of subplasmalemmal regulation of calcium dynamics, and emphasize the importance of store-operated calcium channels during dynamic calcium changes.

Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase.

Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.

Contraction of human hepatic stellate cells activated in culture: A role for voltage-operated calcium channels

Background/Aims: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca 2+] i) and cell contractility. Methods: Binding studies using [ 3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca 2+] i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. Results: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1±1.4×10 4 sites/cell with a Kd of 2.1±0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca 2+] i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca 2+] i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. Conclusion: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca 2+] i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.

Relationship between Intracellular Calcium Store Depletion and Calcium Release-activated Calcium Current in a Mast Cell Line (RBL-1)

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.

The Role of pp60c-src in the Regulation of Calcium Entry via Store-operated Calcium Channels

In many cell types, G protein-coupled receptors stimulate a transient Ca2+ release from internal stores followed by a sustained, capacitative Ca2+ entry, which is mediated by store-operated channels (SOCs). Although it is clear that SOCs are activated by depletion of internal Ca2+ stores, the mechanism for this process is not well understood. Previously, we have reported that inhibitors of tyrosine kinase activity block the bradykinin- and thapsigargin-stimulated Ca2+ entry in fibroblasts, suggesting that a tyrosine kinase activity may be involved in relaying the message from the empty internal Ca2+ stores to the plasma membrane Ca2+ channel (Lee, K.-M., Toscas, K., and Villereal, M. L. (1993) J. Biol. Chem. 268, 9945-9948). We also have demonstrated that bradykinin activates the nonreceptor tyrosine kinase c-src (Lee, K.-M., and Villereal, M. L. (1996) Am. J. Physiol. 270, C1430-C1437). We investigated whether c-src plays a role in the regulation of SOCs by monitoring capacitative Ca2+ entry in 3T3-like embryonic fibroblast lines derived from either wild type or src-/src- (Src-) transgenic mice. We report that Ca2+ entry, following store depletion by either bradykinin or thapsigargin, is dramatically lower in Src- fibroblasts than in wild type fibroblasts. The level of capacitative Ca2+ entry in Src- cells is restored to nearly normal levels by transfecting Src- cells with chicken c-src. These data suggest that c-src may play a major role in the regulation of SOCs.

B cell antigen receptor-evoked calcium influx is enhanced in CD22-deficient B cell lines.

CD22 is a B cell membrane glycoprotein that, upon Ag receptor engagement, becomes rapidly tyrosyl phosphorylated and associates with several signaling molecules including Lyn, Syk, PLCgamma1, and the protein-tyrosine phosphatase, SHP-1. Two allelic forms of murine CD22 exist: CD22.1 is expressed in strains such as NZB and DBA/2, whereas CD22.2 is expressed in BALB/c and most other strains. WEHI-231 cells, which derive from a (BALB/c x NZB)F1 mouse, express one copy of each allele. Previous studies have proposed both positive and negative functions for CD22. We explored the role of CD22 in surface IgM Ag receptor signal transduction by examining signaling in three clonally independent WEHI-231 variants that have lost expression of the CD22.2 allele. This experimental design allowed us to assess the signaling functions of CD22 independent of its developmental role. These variants, which exhibit a 50% reduction of total surface CD22, are hyper-responsive to Ag receptor stimulation: several cellular proteins are hyperphosphorylated on tyrosyl residues and surface IgM-mediated calcium flux is markedly increased. Interestingly, the increased calcium response observed in CD22-deficient cells is due largely to enhanced calcium influx. Reconstitution of CD22 expression reduces these changes. The SHP-1/CD22 association is reduced in CD22-deficient cell lines and is restored by re-expression of CD22. Our results demonstrate that CD22 is a cell autonomous negative regulator of B cell Ag receptor signaling, and suggest that it regulates calcium entry via a mechanism downstream from or independent of calcium release from intracellular stores.

Molecular Biology of Calcium Channels in the Cardiovascular System

Calcium ions are key intracellular messengers in the cardiovascular system. Calcium homeostasis is regulated by an extracellular cycle, which controls the entry and removal of calcium between the cytosol and extracellular space, and an intracellular cycle, which controls calcium fluxes between the cytosol and intracellular stores in the sarcoplasmic reticulum. Several protein families mediate these calcium fluxes including those that (1) regulate the entry of calcium into the cytosol; (2) recognize calcium within the cytosol; and (3) remove calcium from the cytosol. Intracellular calcium binding proteins (the “E-F hand” proteins) recognize the appearance of calcium in the cytosol; in the heart and vascular smooth muscle, these proteins initiate excitation–contraction coupling. Calcium efflux occurs via adenosine triphosphate (ATP)-dependent calcium pumps and sodium–calcium exchangers, while two families of channels—intracellular release calcium channels and plasma membrane calcium channels—regulate calcium entry into the cytosol. The plasma membrane calcium channels, which include the L- and T-type channels, are of the greatest clinical interest because they are targets for pharmacologic therapy. T-type calcium channels, which activate contraction in vascular smooth muscle but have little or no role in cardiac excitation–contraction coupling, appear to be involved in signal transduction pathways that promote cell growth and proliferation. Calcium channel blockers that selectively block T-type calcium channels, therefore, offer a novel approach to cardiovascular drug therapy.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

A novel calcium-sensing domain in the BK channel

The high-conductance Ca2+-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a "core" resembling a subunit from a voltage-dependent K+ channel, and an appended "tail" that plays a role in calcium sensing. Here we present evidence for a site on the tail that interacts with calcium. This site, the "calcium bowl," is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: 1) those that shift the position of the G-V relation a similar amount at all [Ca2+], and 2) those that shift the position of the G-V relation only at low [Ca2+]. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of [Ca2+] or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site.

Increase in [Ca2+]i and subsequent insulin release from beta TC3-cells with the L-type Ca(2+)-channel activator, FPL 64176.

The regulation of calcium entry is central in the control of insulin secretion from the pancreatic β-cell. Glucose is the primary physiological stimulator of insulin secretion and a principal action of glucose is consequently to depolarize the β-cell by closing K+-channels sensitive to the ATP produced during metabolism of the sugar1,2. This will lead to membrane depolarization and activation of the voltage-dependent L-type Ca2+-channels leading to increase in intracellular calcium concentration, [Ca2+]i, and insulin secretion. Pharmacological important secretagogues such as sulphonylureas (e.g. glibenclamide) close the ATP-sensitive K+-channels leading to depolarization of the β-cell membrane, activation of voltage gated Ca2+-channels by opening the channels and initiation of repetitive action potentials. The resulting calcium influx increases the [Ca2+]i and thereby triggers insulin release. Compounds interfering with this process also regulate the insulin secretion and therefore the L-type Ca2+-channel might, like the ATP-sensitive K+-channel, be a suitable target for therapeutic intervention in treatment of Non-Insulin Dependent Diabetes Mellitus (NIDDM). The ligands for L-type Ca2+-channels can be divided into three categories: dihydropyridines, phenylalkylamines and benzothiazepines. Bay K 8644 has become the prototype for the class of dihydropyridines. Some of the dihydropyridines are chiral and resolution of these have revealed the (−)-enantiomers as potent activators, whereas the (+)-enantiomers are weak inactivators3. Analysis of the calcium current activation and deactivation kinetics shows that Bay K 8644 increases the mean open time while it leaves the mean closed times of the Ca2+-channel unchanged4.

Down-regulation or long-term inhibition of protein kinase C (PKC) reduces noradrenaline release evoked via either PKC-dependent or PKC-independent pathways in human SH-SY5Y neuroblastoma cells

Long-term (8–48 h) treatment of SH-SY5Y neuroblastoma cells with phorbol-12,13-dibutyrate (PDBu; 100 nM) promotes the down-regulation of protein kinase C (PKC) subtypes α and ϵ and reduces by up to 60% noradrenaline (NA) release evoked via both PKC-dependent (M 3-muscarinic receptor activation) and PKC-independent (depolarization) pathways, over similar time courses. A similar effect on release is observed following long-term (16–48 h) incubation with the PKC inhibitor Ro 31-7549 (10 μM), even after removal of the inhibitor, indicating a mechanism which is not rapidly reversible. Evidence is presented which suggests that long-term treatment with PDBu does not (1) affect calcium entry, (2) modulate levels of proteins important in the secretory mechanism or (3) reduce the number of secretory vesicles. Thus, the decrease in NA release in SH-SY5Y cells following down-regulation of PKC appears to be the result of a sustained reduction in PKC activity acting on a component of the secretory pathway not involved in the regulation of calcium entry or vesicle number.

CGMP is not required for capacitative Ca 2+ entry in Jurkat T-lymphocytes

In this study, we have addressed the potential role of cGMP in regulating calcium entry in Jurkat T-lymphocytes. These cells display capacitative Ca 2+-entry in response to the intracellular Ca 2+-ATPase inhibitor, thapsigargin (TG). In the presence of extracellular Ca 2+, TG stimulates a sustained elevation of intracellular cGMP levels. In the absence of extracellular Ca 2+, TG induces no apparent increase in the levels of cGMP. However, experiments using Mn 2+, as a surrogate for Ca 2+, demonstrated that TG increased the rate of divalent cation entry in the absence of extracellular Ca 2+. Treatment of Jurkat cells with the guanylyl cyclase inhibitor, LY83583 (20 μM), completely blocked cGMP formation in response to TG. However, LY83583 treated cells still exhibited a sustained, albeit partially reduced, Ca 2+ response induced by TG. These data demonstrate that, in Jurkat cells, the sustained formation of cGMP is dependent on elevated intracellular Ca 2+, and that elevated levels of cGMP are not necessary for the activation of capacitative Ca 2+ entry.

Calcium mobilization by inositol phosphates and other intracellular messengers

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3] is now widely recognized as a messenger controlling the release of calcium from intracellular stores. In oocytes, and also probably in excitable cells, another potential calcium-mobilizing messenger is cyclic ADP ribose, although there is as yet little evidence that its levels are regulated by hormones or other extracellular mediators. In addition to signaling intracellular calcium release, [Ins(1,4,5)P 3] also regulates calcium entry across the plasma membrane, but not in a direct manner. Rather, the depletion of intracellular stores by the calcium-mobilizing action of [Ins (1,4,5)P 3] initiates a process of retrograde signaling whereby the depleted stores generate or release a diffusible messenger that is believed to act on the plasma membrane. A phosphorylated metabolite of [Ins(1,4,5)P 3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P 4], has been proposed to modulate this process, but the literature is not consistent on this point. A recently proposed candidate for the retrograde messenger is an activity extracted from Jurkat cells termed CIF (calcium influx factor), which has many properties consistent with such a messenger. There is also evidence that a GTP-dependent process, possibly involving a small G protein, is involved in signaling calcium entry and may be involved in either the formation or action of the diffusible messenger for calcium entry.

Thapsigargin-induced calcium entry in FRTL-5 cells: possible dependence on phospholipase A2 activation.

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A2 (PLA2) is activated. Since PLA2 is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA2 activity and the entry of calcium. Stimulating 3H-arachidonic acid (3H-AA)-labelled cells with thapsigargin resulted in a substantial release of 3H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA2 activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA2 in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA2 activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca2+ in FRTL-5 cells.

Inhibition of bradykinin- and thapsigargin-induced Ca2+ entry by tyrosine kinase inhibitors

We examined the involvement of tyrosine kinase activity in the bradykinin (BK)-mediated signal transduction process. Immunoblots with anti-phosphotyrosine antibodies following BK stimulation of human fibroblasts showed tyrosine phosphorylation of specific proteins that could be inhibited by the tyrosine kinase inhibitors genistein and tyrphostin. Image analysis data from individual cells stimulated by BK in the presence of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced calcium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. The Ca2+ imaging results were confirmed by 45Ca2+ uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydroxycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that three structurally distinct inhibitors of tyrosine kinase activity inhibited BK-stimulated calcium influx, while an inactive analogue of genistein (daidzein) did not, strongly suggests the involvement of tyrosine kinases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor that lacks endogenous tyrosine kinase activity and is known to be coupled to phosphatidylinositol turnover. We found that calcium entry in HSWP (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also inhibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2+ influx pathway induced by depletion of intracellular calcium stores with thapsigargin also may be regulated via a tyrosine kinase pathway.

Activation and regulation of calcium entry in rat parotid gland acinar cells.

Activation and regulation of calcium entry in rat parotid gland acinar cells.

Ethanol administration in vivo alters calcium ions control in rat striatum

The present paper investigates the effect of chronic ethanol treatment administered through drinking water on [ 3H]nitrendipine binding and 45Ca3uptake in rat striatum. The calcium-independent [ 3H]nitrendipine binding was slightly increased in treated rats, while the calcium stimulation of the binding was reduced to one fifth of the controls. In striatal slices prepared from a similar group of ethanol-treated rats the K +-stimulated 45Ca uptake was greatly reduced. These results are the first evidence of calcium-antagonist binding-site ‘plasticity’ following an in vivo pharmacological manipulation correlated with a change in calcium ion transport. In addition, the effect of ethanol on calcium-entry regulation may be a mechanism important for the understanding of its neurotoxic action.

Transcellular calcium transport by midgut of the blowfly, [formula omitted][formula omitted

Transcellular calcium transport by the internally perfused Calliphora midgut has been measured by simultaneously monitoring 45Ca removal from the perfusing saline (entry to the cells) and its appearance in the bathing saline (exit from the cells). Reduction of the Na + gradient across the basolateral membranes of midgut epithelial cells by removal of bathing Na + or by addition of monensin or ouabain inhibits calcium transport across the basolateral membranes. Calcium entry at the apical membranes is inhibited in parallel. The calmodulin inhibitors, trifluoperazine or calmidazolium, do not directly affect calcium transport nor do they dissociate the parallel changes in calcium entry and exit when calcium exit is inhibited. Experiments with A23187 are consistent with a role for intracellular calcium in regulating calcium entry at the apical membranes. It is suggested that calcium transport out of midgut epithelial cells is largely by Na +-Ca 2+ countertransport, and that entry may be regulated by cytoplasmic calcium so that the calcium influx never exceeds the capacity of the transport mechanisms to pump it out of the cells.

Inhibition by melatonin of dopamine release from rat hypothalamus: regulation of calcium entry

The [Ca 2+] dependency of dopamine release evoked by electrical field stimulation of hypothalamic tissue from female rats at the estrous stage was assessed in the presence of melatonin. At concentrations of 10 −9–10 −6 M, melatonin inhibited the [Ca 2+]-dependent dopamine release. Melatonin reduced tissue uptake of 45Ca 2+ during stimulation either by an electrical field or by elevated K + concentration. About 90% of this inhibition by melatonin of 45Ca 2+ uptake, as well as of dopamine release, was not observed in the presence of the calcium ionophore A23187. Hence, the inhibitory effect on dopamine release by melatonin may stem from the reduction of calcium entry into the presynaptic nerve endings.

Regulation of calcium entry into the extracellular environment of the rat brain

Since neuronal activity is critically related to both intra- and extracellular calcium ion concentrations, there must be a mechanism for regulating the transfer of calcium from the blood into the extracellular environment of the brain. The data presented here support the hypothesis that, within or close to the site of passive permeation of calcium in the choroid plexus, there is a calcium pump capable of recycling the filtered calcium back into the capillary circulation. This pump can be blocked by ruthenium red, a known inhibitor of active calcium transport. The activity of the calcium pump is shown to increase in proportion to the concentration of calcium in the cerebroventricular fluid. Its physiological role therefore would be to insure that the calcium concentration available to the brain will be maintained within a narrow range even in the presence of severe hypercalcemic states.