Differential effects of divalent cations on spontaneous and evoked glycine release from spinal interneurons

The effects of Ca2+, Sr2+, and Ba2+ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the "synaptic bouton" preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40 approximately 50% of control followed by a gradual further decline in mIPSC frequency to approximately 30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+>Ca2+>Sr2+. Moreover, the application of excess external [K+]o solution (30 mM) containing Sr2+ or Ba2+ after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+>or==Ba2+>Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+ was replaced by Sr2+ or Ba2+ (2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+ or Ba2+ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.     

Inhibitory effects of cations on the Ca2+ response of water fibers in the frog tongue.

The effects of various cations on water fibers in the frog (Rana catesbeiana) tongue were investigated. The following results were obtained. 1. Water fibers responded well to Ca2+ and Sr2+. 2. SO2-(4) did not inhibit the Ca2+ response. Cl- at high concentrations had an inhibitory effect on the response to high Ca2+ stimulation. However, when the Ca2+ concentration was low, Cl- did not induce any inhibition. Therefore, low Ca2+ stimulation (0.1 mM CaCl2) was used to examine the inhibitory effect of cation. 3. MgCl2 and MgSO4 strongly inhibited the response to low Ca2+ stimulation. The inhibition by Mg salts was not caused by anion but by Mg2+. 4. The Ca2+ response was inhibited by various cations. The order of the inhibitory effects of cations was as follows: La3+ greater than Mn2+ = Mg2+ greater than or equal to Ni2+ greater than Co2+ greater than monovalent cations (Na+, K+, NH4+ and choline+). 5. The Sr2+ response, as well as the Ca2+ response, was inhibited by Na+ and Mg2+.     

Tension development in frog skeletal muscle induced by silver ions

In single fibers of frog toe muscles placed in a Cl- free MOPS solution containing 1.8 mM Ca2+, tension developed slowly in the presence of very low concentrations of Ag+. This tension was not blocked by the administration of Co2+ or Ni2+. On the other hand, two types of transient tensions developed with the application of 5 microM Ag+, in fibers pretreated with 0-Ca2+ MOPS solution, containing either 2 mM Co2+ or 1mM Ni2+, for 10 min. In the presence of divalent cations or TTX, the first repetitive twitch-like contraction disappeared, indicating this tension is induced by action potentials repeatedly generated by the lack of divalent cations. The 2nd subsequent transient tension was caused by 5 microM Ag+ in the presence of various kinds of divalent cations, or TTX. After reversion to the resting tension, the fiber was contracted by adding more than 0.1 mM of Ca2+ or 25 mM caffeine to the external medium. Even when placed in a Ca2+-free solution containing 3 mM EGTA and 3 mM Mg2+ for 30 min, the fiber still developed an appreciable tension in response to 5 microM Ag+. These findings suggest that a transient development of the Ag+-induced tension does not require the presence of external Ca2+. A specific sulfhydryl reagent, pCMPS, did not contract the muscle fiber. Therefore, Ag+ may develop tension by mediating unknown chemical reaction(s) other than the sulfhydryl group on T-tubular membrane proteins.     

Alpha-latrotoxin and glycerotoxin differ in target specificity and in the mechanism of their neurotransmitter releasing action

alpha-Latrotoxin, a high molecular weight protein (130,000) purified from the venom of the black widow spider, and a partially purified neurotoxin, glycerotoxin, prepared from extracts of the jaw glands of the polichaete annelid Glycera convoluta, were previously found to induce similar effects (stimulation of quantal acetylcholine release) at the frog neuromuscular junction. In the present study parallel experiments performed with these two toxins revealed that only glycerotoxin was able to release acetylcholine from Torpedo electric organ synaptosomes, while alpha-latrotoxin did not affect release in this system. In contrast, alpha-latrotoxin stimulated release of dopamine from PC12 cells (a cloned neurosecretory cell line), whereas glycerotoxin was almost inactive. In rat brain synaptosomes both toxins were active. Preincubation of synaptosomal membranes with glycerotoxin was without effect on the subsequent binding of alpha-latrotoxin. Glycerotoxin application induced depolarization of synaptosomal plasma membrane, massive Ca2+ influx, marked increase of the cytosolic Ca2+ concentration, and stimulation of catecholamine release. The latter effect occurred to the same extent when glycerotoxin was applied either in complete medium (containing both Ca2+ and Mg2+), Ca2+-free medium or divalent cation-free medium. Some of these effects of glycerotoxin in rat brain synaptosomes (depolarization, increased Ca2+ influx and increased cytosolic Ca2+ concentration) resemble effects previously reported for alpha-latrotoxin. However, the secretory response induced by the latter was reduced in Ca2+-free, and abolished in divalent cation-free media. The different target specificity and the lack of binding competition of the two toxins could be due to their ability to recognize different receptors whose distribution overlap only in part in the cellular systems we have studied. The differences in action, on the other hand, could depend on postreceptor events, possibly related to the transmembrane insertion of toxin molecules demonstrated by others in artificial lipid membranes.     

The receptive mechanism of various metallic ions in the lateral-line organ of the tadpoles of Rana catesbeiana.

The stimulating effect on the receptor organ of various mono-and divalent metallic ions on the lateral-line nerve of tadpoles was studied. The orders of the effectiveness were Ag + greater than T1+ greater than K+==Na+ greater than Li for monovalent ions and Sr2+ greater than Mg2+ greater than or equal too Ba2+ greater than Cd2+ greater than or equal too Co2+ greater than Mn2+ greater than Zn2+ for divalent ions. Both agree well with the order of Pearson's softness parameter for monovalent ions and Edwards' modified parameter for divalent ones. All sorts of divalent cations exhibited suppressive effects on the stimulating effect of the monovalent cations when they were applied together with the monovalent cations. A simple selection rule was found for the suppressive effect. The stimulating effect of Na+ or K+ was suppressed by all sorts of divalent cations. But the effect of Ag+ was suppressed by Cd2+, but not by Mg2+ nor by Ca2+. In order to remove the Ag+ effect, a dilute solution of DTT was used, although the effect of K+ or Na+ was removed easily by rinsing with distilled water. From these results the receptive mechanism of the endorgan was thought to be chemical adsorption of ions in the receptor cell membrane. Based on the principle of "hard and soft acid and base", it is suggested that there are two types of binding sites for the ions on the membrane, a soft site and a hard one.     

Effects of cations on Helicobacter pylori urease activity, release, and stability.

The urease of Helicobacter pylori is an important antigen and appears critical for colonization and virulence. Several studies have indicated a superficial localization for the H. pylori urease, and the purpose of this study was to determine the effects of cations on the release and stability of urease activity from H. pylori cells. Incubation of partially purified H. pylori urease in water containing 1, 5, or 10 mM Ca2+, Mg2+, K+, Na+, EDTA, or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] had little effect on activity. In contrast, 1 mM Fe3+, Cu2+, Co2+, or Zn2+ substantially (> 80%) inhibited activity, and 10 mM Fe2+, Mn2+, and Ni2+ inhibited about 30% of the activity. Addition of Ca2+ or Mg2+ markedly decreased extraction of urease from intact H. pylori cells by water, but 1 mM Na+, K+, EGTA, or EDTA each had minimal effects on release, suggesting that divalent cations have a role in attachment of urease to H. pylori cells. The stability of enzymatic activity at 4 degrees C was enhanced by addition of glycerol or 2-mercaptoethanol; however, even after loss of activity, full antigenicity for human serum was retained.     

Effect of divalent cations on adhesion of polymorphonuclear leukocytes to matrix molecules in vitro.

Adhesion of N-formyl-methionyl-leucylphenylalanine-stimulated human polymorphonuclear leukocytes (PMNs) to dishes coated with laminin, fibronectin, or collagen types I and IV was dependent on the presence of magnesium (Mg2+) but not calcium (Ca2+). Addition of manganese (Mn2+) in combination with Ca2+ and Mg2+ further increased the number of PMNs adhering to the matrix proteins. Monoclonal antibody 60.3 (mAb 60.3) was equally effective at inhibiting adhesion of PMNs to all the matrix proteins. The presence of Mn2+ (50 microM), in addition to 1 mM Ca2+ and Mg2+, required higher concentrations of mAb 60.3 to inhibit adhesion of PMNs to collagens type I or IV, suggesting increased affinity of PMNs for these substrates. These findings suggest that the PMNs may regulate the affinity of CD11/CD18 for multiple ligands by binding different divalent cations to the receptor.     

Divalent cation modulation of a-type potassium channels in acutely dissociated central neurons from wide-type and mutant Drosophila

Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K+ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd2+ enhanced the peak inactivating current (I(A)) without affecting the delayed component (I(K)). The same results were obtained in Ca(2+)-free external solution, and from slo1 mutation, which eliminates transient Ca(2+)-activated K+ current. Micromolar Cd2+ and Zn2+, and millimolar Ca2+ and Mg2+ all shifted the steady-state inactivation curve of I(A) without affecting the voltage-dependence of I(A) activation, whereas millimolar Cd2+ markedly affected both the activation and steady-state inactivation curves for I(A). Divalent cations affected I(A) with different potency; the sequence was: Zn2+ > Cd2+ > Ca2+ > Mg2+. The modulation of I(A) by Cd2+ was partially inhibited in Sh(M), a null Shaker (one of I(A)-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on I(A) activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K+ channels through specific binding to extracellular sites of the channels.     

Effects of divalent cations and saccharides on Vibrio metschnikovii cytolysin-induced hemolysis of rabbit erythrocytes.

Divalent cations and polysaccharides such as inulin and dextran reversibly inhibited hemolysis of rabbit erythrocytes caused by Vibrio metschnikovii cytolysin. On the basis of the 50% inhibitory doses, the cations were divided into two groups, group I (Cd2+, Cu2+, Ni2+, Sn2+, and Zn2+) and group II (Ba2+, Ca2+, Co2+, Mg2+, Mn2+, and Sr2+). Neither divalent cations nor polysaccharides interfered with the binding of toxins to the erythrocyte membrane. Group I cations disturbed tetramer formation of cytolysin on the cytolysin-lysed erythrocyte membrane, although group II cations and dextran did not affect the process. Erythrocytes treated with cytolysin in the presence of group II cations or dextran lysed after transfer to toxin- and inhibitor (group II cations or dextran)-free buffer at both 37 and 4 degrees C. However, erythrocytes treated in the presence of group I cations lysed at 37 degrees C but not at 4 degrees C, indicating that group I cations block the temperature-dependent lesion (tetramer)-forming step subsequent to the binding of cytolysin to the erythrocytes. The cytolysin-treated erythrocytes swelled in a colloid osmotic manner, and the swelling was preceded by the binding and the lesion-forming steps. It is also suggested that the lysis of the erythrocytes proceeds in a temperature-independent manner and that the cytolysin does not bind to the erythrocytes at 4 degrees C. These findings suggest that the sequence of V. metschnikovii cytolysin-induced hemolysis is defined by three steps: (i) a temperature-dependent binding step, (ii) a temperature-dependent lesion-forming step, and (iii) a temperature-independent lysis step.     

Effects of divalent cations on biphasic potassium contractures and on contractile inactivation in low calcium solutions in frog single twitch muscle fibers

When the concentration of external Ca2+ was reduced for 30 sec in a single twitch muscle fiber of a frog, the peak tension of the initial component of biphasic 80 mM K+ contractures was potentiated, whereas that of the secondary component was markedly inhibited, despite the fact that in the early stage it was potentiated usually in case of contractures in 60 and 40 nM K+ but rarely in 80 mM K+. These changes were not observed, however, in the presence of 0.5-1 nM Mn2+, i.e., Mn2+ having been substituted for Ca2+. The foregoing result and the authors' previously reported data indicated the following. First, the concentrations of divalent cations having an equal effect in bringing about the peak tensions of both components are 3 mM Mg2+, 0.7 mM Mn2+, 0.5 mM Ni2+, and 1.8 mM Ca2+. Secondly, this sequence constitutes their increasing order of effectiveness on the time course of the secondary component. Meanwhile, a similar order was found to exist in another experiment concerning the effectiveness in inhibiting the inactivation of K contractures facilitated by lowering the concentration of external Ca2+. All these findings indicated that the divalent cations act on the activation processes of both components in a stabilizing manner, differing from the way in which they act on the inactivation process of the secondary component. The mechanisms in which the peak tensions of the initial and secondary components are inhibited in a low Ca2+ solution and the divalent cations act on both components are discussed. Finally, another experiment made in the absence and presence of Ca2+ revealed that the effect of high concentrations of Mn2+ in the initial component is different from that on the secondary component.     

Modulation of fetal and adult acetylcholine receptors by Ca2+ and Mg2+ at developing mouse end-plates

It has long been known that extracellular Ca2+ and Mg2+ modulate synaptic transmission at the neuromuscular junction, acting both pre- and post-synaptically. Relevant questions concerning the modulation of acetylcholine (ACh) receptors (AChRs) are however still open: are the fetal (gamma-AChR) and adult (epsilon-AChR) receptors modulated differently? Does the ACh concentration influence the effect of divalent cations? Is the effect on channel open duration dependent on type and concentration of divalent cation? These questions were addressed by studying the modulation of the single-channel behaviour of gamma- and epsilon-AChRs by Ca2+ and Mg2+ at the endplate of muscle fibres acutely dissociated from 12- to 14-day-old mice. Ca2+ reduced the conductances of the two receptor channels comparably. Mg2+ had a stronger effect than Ca2+ and reduced the conductance of epsilon-AChR significantly more than that of gamma-AChR. With 0.1 microM ACh, Ca2+ and Mg2+ increased the mean open duration of gamma- and epsilon-AChR channels comparably. At 100 microM ACh, gamma- and epsilon-AChR channels opened in bursts of strikingly similar duration, which was unaffected by divalent cations. These findings indicate that Ca2+, and even more so Mg2+, may regulate synaptic transmission by modulating the function of AChRs in addition to the well-established effects on transmitter release.     

Effects of divalent cations and of catecholamines on the late response of the superior cervical ganglion.

The characteristics of the late response of the superior cervical ganglion of dogs were studied by close-arterial injection of catecholamines and divalent cations to the ganglion. 2. Dopamine, noradrenaline and adrenaline inhibit the late response as well as ganglionic activity induced by other means. The effect of dopamine is brief but that of adrenaline is prolonged. 3. Cd2+, Co2+, Ni2+, Zn2+, Hg2+ and Fe2+ markedly potentiate the late response, whereas Mn2+, Ca2+ and Mg2+ inhibit it. 4. may potentiate ganglionic activity triggered by other ganglionic stimulants. The Cd-augmented activity may be blocked by a ganglion-blocker which is specific to the stimulant. 5. CdCl2 may exhibit a direct ganglion-stimulating action on a ganglion which shows prominent late responses and has been conditioned by tetanic preganglionic stimulation. 6. CdCl2 and MnCl2 may inhibit ganglionic transmission by suppressing acetylcholine release from presynaptic nerve terminals. 7. It is concluded that the late response represents the late discharges of ganglion cells, which are very sensitive to inhibition by CaCl2 and MnCl2 and may be potentiated by CdCl2.     

Regulation of PTH receptor-adenylate cyclase system of canine kidney: influence of Mn2+ on effects of Ca2+, PTH, and GTP

Metal ions play important roles in the regulation of the activation of adenylate cyclase. Previous studies have suggested that an important site of action of metal ions is at or closely related to the nucleotide regulatory protein. The present studies examine the nature of the regulation of enzyme activity by divalent cations and the influence of Mn2+ on hormone binding and stimulation of adenylate cyclase. Studies were performed in canine renal cortical membranes. Substitution of Mg2+ by Mn2+ was associated with a progressive decline in the ability of GTP or PTH to stimulate adenylate cyclase activity. Mn2+ did not alter specific binding of an iodinated PTH analogue. However, in spite of the loss of guanine nucleotide stimulation of enzyme activity, the effects of guanine nucleotide on PTH binding were not altered in the presence of Mn2+. Substitution of Mg2+ by Mn2+ abolished the inhibitory effect of Ca2+ on basal adenylate cyclase activity. Similarly, the effects of GTP or PTH to enhance the inhibitory effects of Ca2+ on enzyme activity were abolished in the presence of Mn2+. Since Mg2+ and Ca2+ compete for a common allosteric site and Mn2+ abolished the effects of these cations, it would appear that Mn2+ also competes for the binding site of Mg2+ and Ca2+. The present studies demonstrating that Mn2+ does not affect hormone binding or the actions of guanine nucleotides on hormone binding yet totally eliminates the effect of GTP on enzyme activity indicate that the effect of Mn2+ occurs at the level of the interactions of the nucleotide regulatory component with the catalytic unit. In addition, these data suggest that there are two functionally distinct sites of guanine nucleotides with different ionic requirements.     

Effect of metal ions on diphtheria toxin production.

The effect of several metal ions on the production of diphtheria toxin was tested. By using the gel immunodiffusion system for detecting toxin, a wide range of metal ion concentrations was conveniently surveyed. Five divalent cations, Fe2+, Cu2+, Co2+, Ni2+, and Mn2+ inhibited toxin production within a range of concentrations that did not inhibit growth of the producing strain. Growth and toxin production were inhibited at identical concentrations by both Cd2+ and Zn2+, whereas Al3+ and Sr2+ affected neither growth nor toxin production over the range of concentrations tested. The data showed that Fe2+ was the most effective inhibitor on an equivalence basis, followed by Cu2+, Co2+, and Ni2+ in descending order. All eight strains of Corynebacterium diphtheriae chosen from diverse ecological origins responded similarly to all metals at similar concentrations. A mutant strain which produces toxin at Fe2+ concentrations 500 times greater than are inhibitory for the parent strain had simultaneously acquired resistance to inhibitory concentrations of Cu2+, Co2+, Ni2+, and Mn2+. This suggests that there is at least one common point in the activity of all these metal ions, and that toxin may respond broadly to changes in metal ion concentrations in the environment.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

Effects of Bay K 8644 on spontaneous and evoked transmitter release at the mouse neuromuscular junction

The dihydropyridine, Bay K 8644, was applied in vitro to mouse phrenic nerve-diaphragm muscle preparations. The drug increased both spontaneous and evoked release of acetylcholine from the motor nerve terminal in a concentration- and time-dependent manner. The rise in miniature endplate potential frequency, however, was the result of an increased intraterminal mobilization of free calcium, rather than well-established activation of voltage-dependent calcium channels. This view is supported by the following observations: (1) an increase in frequency was apparent in Ca2+-free medium; (2) Bay K 8644 is known to require a moderate depolarization to affect Ca2+ channels, but no membrane depolarization was detected; and (3) exposure to low Ca2+ and high Mg2+ medium did not diminish the effect on miniature endplate potential frequency. In a medium containing low Ca2+ and high Mg2+, Bay K 8644 increased quantal content of the evoked endplate potentials to a greater degree and with a faster time course than the frequency of miniature endplate potentials. This enhancement in evoked release did not appear to be caused solely by an increase in cytoplasmic Ca2+, but rather reflected at least in part the Bay K 8644-induced activation of voltage-gated Ca2+ channels, perhaps L-type, at the presynaptic nerve terminal. Thus, we propose that Bay K 8644 exerts dual effects on the motor nerve endings, characterized by a primary action on the presynaptic Ca2+ channels and a secondary action associated with the elevation of intracellular Ca2+ concentration.     

High affinity binding of the calcium channel blocker, (+)-[methyl-3H]PN200-110(3HPN) in rat brain

PN200-110 is a recently introduced 1,4-dihydropyridine which has been demonstrated to be a potent calcium channel blocker. 3HPN has been shown to bind in a specific saturable manner to P2 fractions obtained from brain homogenates from male Sprague-Dawley rats. 3HPN binding was found to be temperature-dependent. Specific 3HPN binding was maximal at 25 degrees C; binding decreased at 2 degrees C and 37 degrees C. The KD calculated from Scatchard analysis was 0.0943 +/- 0.0038 nM while the Bmax was found to be 109.1 +/- 2.3 fmol/mg protein. A concentration dependent inhibition of 3HPN binding by various cations was determined and found to be as follows: ZN2+ greater than La3+ greater than Rh3+, Al3+ greater than Co2+, Ni2+, Mn2+ greater than Ca2+, Mg2+ greater than Ba2+ greater than Sr2+. These results provide evidence for the existence of central high affinity dihydropyridine receptor sites in rat brain.     

Uncoupling cation effects on cardiac contractility and sarcolemmal Ca2+ binding.

Studies have been performed correlating the effects of cationic uncouplers on intact cardiac muscle and on Ca2+ bound to isolated cardiac plasma membranes. Sarcolemmal vesicles were isolated from neonatal rat hearts. Ca2+ binding experiments were performed and Scatchard plot analysis indicated two classes of Ca2+ binding sites. The ability of certain cations to displace Ca2+ bound to these vesicles was measured. The effects of these same cations on the tension developed by neonatal rat papillary muscles and on the Ca2+ content of tissue culture cells (from neonatal rat heart) were measured. The results show that a) the selectivity sequence (Y3+ greater than Nd3+ greater than La3+, Cd2+ greater than Co2+ greater than Mg2+) of sarcolemmal Ca2+ binding sites is the same as the effective uncoupling sequence; b) the amount of Ca2+ bound at these sites (approximately 700 micromol/kg wet wt) is more than adequate to support tension development; c) the dependence of sarcolemmal Ca2+ binding at low-affinity sites and tension development on [Ca2+]o is essentially the same. It is then reasonable to propose that the Ca2+ bound to these sarcolemmal sites plays an important role in controlling the amount of Ca2+ available to the myofilaments and thus myocardial contractility.     

The ionic basis of chemotaxis. Separate cation requirements for neutrophil orientation and locomotion in a gradient of chemotactic peptide.

The behavior of cells undergoing chemotaxis may be analyzed in terms of their orientation, a static characteristic, and of their locomotion. We have examined the extracellular divalent cation requirements for orientation and locomotion of rabbit polymorphonuclear leukocytes (neutrophils) in a gradient of the chemotactic peptide N-formyl-methionyl-leu-cyl-phenylalanine (F-Met-Leu-Phe) using the chemotaxis chamber recently developed by Zigmond. This chamber allows direct observation of cells attached to glass coverslips as they move up a gradient of chemotactic agent established across a 1-mm bridge. The orientation of neutrophils in the direction of the gradient was equally efficient whether cells and F-Met-Leu-Phe were suspended in merium supplemented with both Ca2+ and Mg2+ (complete medium), with Mg2+ but not Ca2+ (by simple omission of Ca2+ or by addition of EGTA), or with nonsupplemented medium (by omission of Ca2+ and Mg2+ or by addition of EDTA). These data confirm and extend Zigmond's earlier observation that exogenous divalent cations are not required for polymorphonuclear leukocyte orientation toward the chemotactic peptide. In contrast, cell locomotion, determined by linking the chemotaxis chamber to a time-lapse videocassette recorder and TV monitor, is markedly affected by the medium's content of divalent cations. Cells suspended in medium supplemented with Mg2+ but not calcium (by omission or chelation) or in nonsupplemented medium moved on the average 25% more rapidly than cells in complete Ca2+ and Mg2+ medium. Although the simple omission of Mg2+ does not prevent chemotaxis, chelation of Mg2+ in the medium completely abolishes leukocyte locomotion. Addition of varying concentrations of Mg2+ to the buffer in the presence of EDTA established that cell movement is fully restored by Mg2+ concentrations in the range of 3 X 10(-9) M, concentrations easily attained in the absence of added Mg2+. It was concluded that neither Ca2+ nor Mg2+ is needed for orientation in response to F-Met-Leu-Phe. However, low levels of exogenous Mg2+ but not Ca2+ are required for effective locomotion of neutrophils in the Zigmond changer. This result contrasts with data obtained in the Boyden chamber, where exogenous Ca2+ is considered essential for maximum chemotactic response.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.