Removal Of External Calcium Research Articles

Properties of the residual calcium pools in human red cells exposed to transient calcium loads.

1. Inosine-fed human red cells, pre-loaded with calcium with the use of the ionophore A23187, and reincubated at 37 degrees C after ionophore and external calcium removal, pumped calcium out initially fast and then substantially slower when the residual calcium was still very high (García-Sancho & Lew, 1988 a, b). Particularly surprising was the finding of such residual calcium pools in a subpopulation of cells (L cells, García-Sancho & Lew, 1988b) with normal ATP, high active calcium pumping rates, and K+ channels which remained inactive during the density separation procedure (SCN- treatment; García-Sancho & Lew, 1988a). The purpose of the present experiments was to investigate the properties and possible origins of the residual calcium pools. 2. At each ionophore concentration, the pool size increased with the duration and magnitude of the calcium load. At comparable calcium loads, the pool size was smaller the higher the ionophore concentration used. 3. In cells from the same sample, residual calcium pools were present in cells that became dense after SCN- treatment (H cells) as well as those that remained in the light-cell fraction (L cells). Residual calcium was always higher in H cells than in L cells. 4. Re-exposure of cells to ionophore in Ca2+-free media could rapidly extract over 99% of their residual calcium. Residual calcium is therefore in a rapidly mobilizable form within a membrane-bound compartment. 5. Iodoacetamide-induced ATP depletion of H cells with high residual calcium-stimulated calcium loss. Such stimulation could only occur if ATP depletion inhibited a calcium-retaining process within the cells. 6. L cells with high residual calcium may have failed to dehydrate during SCN- treatment because of irreversible K+ channel inactivation or because K+ permeabilization would no longer generate a dehydrating net cation loss. These possibilities were tested and ruled out since it was found that virtually all cells became dense in low-K+, SCN- media after valinomycin addition or re-exposure to A23187 + calcium. 7. The results suggest that part if not all of the residual calcium is contained within compartments with the properties of endocytic inside-out vesicles capable of ATP-dependent calcium accumulation, such as those found in normal and abnormal human red cells (Lew, Hockaday, Sepúlveda, Somlyo, Somlyo, Ortiz & Bookchin, 1985).

Comparative Effect of Angiotensin II, Potassium, Adrenocorticotropin, and Cyclic Adenosine 3′,5′-Monophosphate on Cytosolic Calcium in Rat Adrenal Cells

The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells.

Calcium and osmotic stimulation in renin release from isolated rat glomeruli.

The effects of changes in osmolality and calcium concentration on renin release (RR) from isolated superfused rat glomeruli were studied. The undisturbed RR followed a first order fall with a half-time of about 100 min (n = 45). Changes in the osmolality between 270 and 350 mOsm/kg resulted in dose-dependent changes in the RR rates. Hypoosmotic treatment stimulated the RR transiently, whereas hyperosmotic treatment produced a sustained inhibition. The dose-response relationship was log-linear between 270 and 320 mOsm/kg. A decrease in osmolality of 20 mOsm/kg gave proportional increases in RR irrespectively of the RR rate preceding the stimulus. Removal of calcium stimulated the RR by 10 times (n = 5, p less than 0.001) and a subsequent decrease in osmolality of 20 mOsm/kg stimulated the RR proportionally to that observed in the series containing 2 mM calcium. A decrease in osmolality was able to stimulate RR (n = 5.5, p less than 0.05) even when the calcium concentration in the medium was simultaneously raised from 0 to 2 mM. A hyperosmotic Ringer (+ 300 mOsm/kg), inhibited RR to very low levels. A subsequent removal of external calcium was now unable to stimulate the release (n = 5.5). In a less hyperosmotic Ringer (+ 50 mOsm), the RR was inhibited, but a removal of external calcium now stimulated RR. It is suggested that the osmosensitivity of the RR process reflects a waterflux-driven fusion of secretory granules with the cell membrane, and that calcium affects an intragranular equilibrium between aggregated, osmotically inert granule content and dissolved, osmotically active granule content.

Indirect adrenergic effect of compound 48/80 in cat cerebral arteries.

Compound 48/80 evoked an increase in the spontaneous tritium release from cat cerebral arteries prelabelled with 3H-NA. This increase was abolished after cervical gangliectomy, external calcium removal or in the presence of colchicine. Cocaine brought about an enhancement in the release of radioactivity elicited by compound 48/80 while diphenhydramine did not affect it. These results suggest that compound 48/80 has an indirect adrenergic effect in cat cerebral arteries which is not mediated by the release of histamine from mast cells.

Indirect adrenergic effect of histamine in cat right atrium

1. 1. Superfused cat atrial fragments preincubated with [ 3H]noradrenaline showed an increased tritium release under the action of 10 −4 M histamine. 2. 2. Reserpine pretreatment of the animals, external calcium removal or the presence of 10 −6 M cocaine, 10 −6 M diphenhydramine or 10 −3 M colchicine in the superfusion medium significantly reduced this effect of histamine (10 −4 M). 3. 3. Diphenhydramine (10 −5 M) also decreased the release of tritium brought about by 10 −6 M tyramine. 4. 4. These results suggest that histamine may release neurotransmitter from cardiac sympathetic nerve endings by means of an exocytotic process and needs to enter the nerve terminals through the amine uptake system.

Some effects of removal of external calcium on pig striated muscle.

Bundles of about 800 cells from the m. thyreopharyngicus of pigs were used to measure activation and inactivation during contracture by K+ depolarization. When [Ca2+] in the medium was lowered to less than 5 X 10(-10) M for 3 min (replacing Ca2+ by Mg2+) the activation occurred at the same [K+] in the medium as in normal solution (3 mM-Ca2+) but inactivation was shifted to lower external [K+]. The absolute value of this shift in terms of membrane potential is uncertain, because [K+] at the cell surface is unknown. Exposure for 4 min to Ca2+-free medium (Ca2+ being replaced by Mg2+) had no effect on contractility tested after a subsequent rest of 22-25 min in normal solution ( [Ca2+] = 2 mM). However, if the muscle underwent one maximal K+ contracture in Ca2+-free medium the response (tetanus or K+ contracture) after the same interval in normal solution was strongly reduced, although the membrane potential recovered fully. K+ contractures in normal solution could be repeated without loss of contractile force. A K+ contracture in Ca2+-free medium had very little effect on the response to caffeine, tested after 25 min in normal solution. It seems that Ca2+ is lost into Ca2+-free medium only during depolarization, from a site which is not accessible to Ca2+ from outside at the resting membrane potential, or from inside at any membrane potential. This site might be located inside the transverse-tubular membrane and, when loaded with Ca2+, might represent the positive group of the model of Chandler, Rakowski & Schneider ( 1976b ), the movement of which during depolarization activates and inactivates the Ca2+ release from the sarcoplasmic reticulum.

Brain microvessel endothelial cells in tissue culture: a model for study of blood-brain barrier permeability.

Endothelial cells were prepared from bovine microvessels and grown in tissue culture. They contained factor VIII/von Willebrand antigen, the most specific market available for determination of the endothelial origin of cells in culture. The cultured cells formed complex tight junctions and contained few pinocytotic vessels. These properties are responsible for formation of the blood-brain barrier in vivo. When monolayers of the endothelial cells were exposed briefly to a calcium-free solution or treated with 1.6 M arabinose, distinctive morphological changes occurred in the intercellular contacts. In either case, a normal structure was reestablished following return to control medium. To assess the effect of these treatments on transcellular permeability, we measured the movement of sucrose labeled with carbon 14 across a monolayer of endothelial cells cultured on a collagen-coated nylon mesh. Removal of external calcium increased the rate of sucrose movement by 120%; the arabinose treatment increased transcellular flux by 40%.

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na +-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH i is more acidic than the external medium (pH o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na + to sperm suspended in Na +-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH i by 0.4–0.5 pH units. That this pH i change is the causal trigger of motility was suggested by experiments using NH 4Cl and nigericin, which increased the pH i and resulted in activation of motility in the absence of Na +. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH i was observed in one species of sea urchin ( L. pictus) but not in the other ( S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca 2+ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca 2+ uptake. Either mitochondrial agents or the removal of external Ca 2+ could also block this pH i change suggesting a similar mechanism is involved.

UREA PARTHENOGENETICALLY ACTIVATES THE CORTICAL REACTION AND ELONGATION OF MICROVILLI IN EGGS OF THE SEA URCHIN,STRONGYLOCENTROTUS PURPURATUS

Isotonic urea is believed to activate sea urchin eggs by triggering event(s) that normally follow cortical granule secretion at fertilization, particularly surface perturbations that result in elongation of microvilli (Mazia et al., 1975). However, Moser (1940) reported that urea triggered the cortical reaction. Transmission electron microscopy showed that unfertilized Strongylocentrotus purpuratus eggs discharge their cortical granules in isotonic urea (containing 1.0 to 0.1 mM CaCl2 or 25 mM EGTA) to form incipient fertilization envelopes and hyaline layers. These investments quickly disperse in urea. Elongation of microvilli follows cortical granule discharge. Urea-activated eggs can be fertilized after return to sea water and fail to elevate fertilization envelopes but do form hyaline layers. Hyalin must be secreted from a secondary reservoir in these eggs, since the cortical granule store is discharged during the prior urea activation. Cortical granule secretion and elongation of microvilli do not occur in urea plus 10 mMCaCl2. These eggs form normal fertilization envelopes and hyaline layers when fertilized after return to sea water. Our results show that: (1) urea triggers an early event in sea urchin egg activation that stimulates cortical granule secretion; (2) cortical granule discharge precedes elongation of microvilli in urea-activated eggs as it does during normal fertilization; and (3) reduction or removal of external calcium is required for activation by urea.

The role of calcium in the control of renin release.

The effects of removing external calcium and inhibiting entry of calcium into the cell by treatment with D-600 on renin release from renal cortical slices of male Sprague-Dawley rats were examined. Baseline renin release, angiotensin II (AII)-induced inhibition, and isoproterenol-induced stimulation of renin release were studied. Removal of external calcium by chelation with 5 mM EGTA inhibited basal renin release while treatment with 1 mM EGTA stimulated basal renin release slightly. Incubation of slices with zero calcium medium containing 1 mM EGTA had no effect on isoproterenol-induced stimulation of renin release. In contrast, similar treatment reduced the inhibitory effect of AII from 58.7% of baseline to 85.3% (p less than 0.001). Similarly, blockage of calcium entry into cells with D-600 had no effect on isoproterenol-induced stimulation of renin release but abolished AII-induced inhibition. Replacement of sodium in the bathing medium with choline had no effect on baseline renin release or on AII-induced inhibition of renin release, ruling out the possibility that D-600 altered renin release via an effect on sodium influx. Taken together, the data strongly suggest that AII-induced inhibition of renin release is partially dependent on the presence of external calcium but that isoproterenol-induced stimulation of renin release is not.

Pepsinogen release from isolated gastric glands.

The in vitro release of pepsinogen was studied using a preparation of isolated gastric glands from rabbits. The pepsinogen content of the glands was estimated to be about 700 U/mg dry wt. Spontaneous release of pepsinogen was found to be less than 1% of the total per hour and relatively constant for at least 2 h. Pepsinogen release was stimulated in a dose-dependent manner by both carbachol and isoproterenol. The cholinergic and beta-adrenergic stimulation was selectively inhibited by atropine and propranolol, respectively. Removal of external calcium inhibited the responses to both isoproterenol (partially) and carbachol (completely). Several agents, including histamine, prostaglandin (E2), and synthetic secretin, were found not to stimulate pepsinogen release. However, a crude secretin preparation (Boots) was found to produce significant stimulation. Dibutyryl cAMP increased pepsinogen release in a dose-dependent manner. Isoproterenol was found to increase the cAMP content of gastric glands and to stimulate adenylyl cyclase activity in homogenates. The beta-adrenergic stimulation of adenylyl cyclase was found to be selective for a population of gastric cells that was relatively depleted of parietal cells and distinct from the histamine-stimulated adenylyl cyclase activity. The results indicate that pepsinogen secretion by the gastric chief cell is regulated, in part, by separate cholinergic and beta-adrenergic mechanisms and that both calcium and cAMP play a role in this regulation.

The effect of ethanol on inhibitory and motor responses in the rat and rabbit anococcygeus and the bovine retractor penis muscles.

1 Ethanol (200 mM) reduced the response to inhibitory nerve stimulation in the rat and rabbit anococcygeus and the bovine retractor penis (BRP) muscles. Ethanol also reduced the response to the inhibitory extract from the BRP consistent with the inhibitory factor in these extracts playing some part in the response to inhibitory nerve stimulation. 2 Ethanol's effect on the response to other inhibitory stimuli was examined in the rabbit anococcygeus and the BRP. In the anococcygeus the response to carbachol was reduced, to bradykinin and isobutylmethylxanthine (IBMX) unaltered, and to isoprenaline and adenosine 5'-triphosphate (ATP) potentiated. In the BRP responses to IBMX and sodium nitroprusside were unaltered but in this tissue the response to isoprenaline was reduced. Ethanol's ability to reduce inhibitory responses is, therefore, selective and confined to inhibitory nerve stimulation, inhibitory extract, carbachol, and, in the BRP, isoprenaline. 3 Ethanol reduced the rate of development of inhibition even where the magnitude of the inhibitory response was unaltered. 4 In the rat anococcygeus, ethanol (200 mM) potentiated the response to motor nerve stimulation and to noradrenaline (NA) at low frequencies and low concentrations respectively. Higher ethanol concentrations (400 mM) reduced the response to both motor nerve stimulation and NA. The motor response to carbachol was also reduced. 5 Ethanol (200 mM) itself caused an easily reversible contraction in all three tissues. This was not due to the release of NA but was highly sensitive to the removal of external calcium from the medium. 6 A unified explanation of these varied effects of ethanol based on a reduction in membrane binding of calcium and a reduced efficiency of receptor coupling is suggested.

Phosphorylation of the same specific protein during amylase release evoked by beta-adrenergic or cholinergic agonists in rat and mouse parotid glands.

Stimulation of amylase secretion from the rat parotid gland by beta-adrenergic agonists is associated with a specific phosphorylation of three membrane-bound proteins designated as proteins I, II, and III [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. In contrast, stimuliation by carbachol induced significant phosphorylation of only protein I. This phosphorylation was low compared to isoproterenol-induced phosphorylation but corresponded to the smaller enhancement of amylase secretion. The mouse organ, however, is almost equally sensitive to beta-adrenergic and to cholinergic agonists. Incubation of mouse parotid gland slices with either 20 microM isoproterenol or 10 microM carbachol resulted in strong and comparable releases of amylase, which were accompanied by comparable phosphorylations of protein I. Proteins II and III were phosphorylated only in the presence of isoproterenol. Removal of external calcium by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate abolished the carbachol-induced release of amylase but not the phosphorylation of protein I. Isoproterenol-induced secretion of amylase and phosphorylation of proteins I, II, and III were not inhibited under these conditions. Amylase release stimulated by the ionophore A-23187 was accompanied by the phosphorylation of protein I. Two-dimensional electrophoresis revealed that the radioactive spot corresponding to protein I was located at the same position after cholinergic and after beta-adrenergic stimulation, indicating that both stimuli led to the phosphorylation of the same membrane-associated protein. These findings strongly support the view that the phosphorylation of protein I is an important step in the sequence of events leading from receptor activation to exocytosis.

Activation of tryptophan hydroxylase from slices of rat brain stem incubated with agents which promote calcium uptake or intraneuronal release

Incubation of slices of rat brain stem under conditions that promote calcium accumulation by nerve tissue (Na-free medium, ouabain. A23187) or intraneuronal release of calcium from mitochondrial stores (metabolic inhibitors, cyanide, azide, rotenone, guanidine and dicoumarol) has been found to result in an increase in the activity of tryptophan hydroxylase, prepared from the slice preparations in a low speed supernatant fraction and assayed in the presence of 200 μM L-tryptophan and 50 μM DL-6-methyl-5,6,7,8-tetrahydropterin (6-MPH 4). The increase in enzyme activity following treatment of the slices with Na +-free medium, ouabain, ionophore A23187, guanidine, cyanide or azide was reflected in altered kinetic properties of the enzyme, namely a decrease in the K m of the enzyme for both substrate and artificial reduced pterin cofactor. In addition, a modest increase in V max was observed, but was not always statistically significant. The alterations in kinetic properties obtained following the different treatments to the slice preparations were similar to those obtained with enzyme prepared from brain stem slices depolarized in a potassium-enriched incubation medium. This depolarization-induced activation of tryptophan hydroxylase is a calcium-dependent phenomenon. In agreement with this, no increase in enzyme activity was observed when calcium ions were omitted from the Na +-free medium and media containing ouabain or A23187. Removal of external calcium did not abolish the increase in enzyme activity obtained with metabolic inhibitors, presumably because these substances release calcium from mitochondria. The data are consistent with the view that a rise in free intraneuronal calcium triggers certain biochemical events which activate tryptophan hydroxylase.

Measurement of calcium influx under voltage clamp in molluscan neurones using the metallochromic dye arsenazo III.

1. The metallochromic indicator dye, arsenazo III, was injected into somata of molluscan neurones from Archidoris monteryensis. Membrane current and dye absorbance change were simultaneously monitored under voltage clamp. 2. Absorbance measured at 660 nm increased during positive-going voltage steps large enough to activate membrane conductances. In situ idifference spectra were qualitatively similar to dye, dye-calcium difference spectra recorded in vitro. The absorbance change was abolished by either a thorough removal of external calcium or internal chelation of calcium by EGTA. It was concluded that the absorbance increase primarily reflected changing internal calcium concentration and that the calcium entered from the outside. 3. Dye absorbance increased in a nearly linear fashion during voltage clamp pulses of 100--300 msec duration. This is in qualitative agreement with electrical stuidies which demonstrated only fractional inactivation of calcium conductance during such periods. Plots of absorbance change vs. Vm peaked at +30 to +40 mV and fell off sharply until approximately +70 mV where the slope became less steep. A null or reversal of the absorbance change was generally observed aroung +110 mV. Evidence is presented that calcium influx was in some cases sufficient to cause sizeable changes in its equilibrium potential. 4. During multisecond voltage clamps the slope of the absorbance change showed a large decline. Where barium substituted for calcium as the influx species in identical clamps, the absorbance at 660 nm also increased but in a much more linear fashion. Except for a slight effect on the initial few pulses, the absorbance signal did not recover after a period of barium influx. These results suggest that part of the slope decline might result from processes related to calcium uptake and not to membrane conductance decrease. 5. Dye absorbance changes during normal and TEA action potentials were measured. Comparison of these changes with voltage clamp records indicated that calcium influx during a spike was capable of raising concentration by roughly 2 X 10(-7) M if the cell were considered to be a uniform sphere with no buffering capacity. Calcium influx during action potentials was increased dramatically by TEA, primarily as a result of a prolonged plateau phase. The existence and duration of the plateau was controlled mainly by potassium conductance systems, however. There was no evidence found for facilitation of the calcium conductance. 6. Following a moderate influx of calcium it required 20--60 sec, depending on the neurone, for the dye absorbance to return to base line (at 9 degrees C). The recovery time course showed a marked difference when examined at different wavelengths. For lambda = 660 nm there was an initial period in which the absorbance decreased rapidly, followed by a slower phase which generally carried the absorbance below the initial (prepulse) value...

Endogenous prostaglandin E and contractile activity of isolated ileal smooth muscle.

The effects of endogenous prostaglandin E (PGE) on the contractile activity of isolated cat ileal muscle rings were studied. Force development in circularly oriented muscle fibers were recorded. The muscles contained a mean basal PGE concentration of 1 +/- 0.6 ng PGE per g wet wt (mean +/- SE) as measured by radioimmunoassay. Acetylcholine (ACh) or elevated potassium caused contractions and enhanced PGE concentration (P less than 0.05). Removing Ca2+ from high potassium solutions blocked contraction, but PGE concentration still increased (P less than 0.02). The prostaglandin synthetase inhibitors, indomethacin and 5,8,11,14-eicosatetraenoic acid (ETA) reduced PGE concentration in muscles (less than 50 pg PGE per g muscle) and increased the magnitude of contractions induced by either ACh or elevated potassium. Spontaneous contractions were observed in many tissues after inhibition of PGE synthesis. In conclusion, endogenous PGE limits spontaneous and depolarization-induced muscular activity in cat ileal circular muscle. Synthesis of PGE was increased by depolarizing stimuli whether or not contractions were blocked by the removal of external calcium.

An analysis of the actions of prostaglandin E1 on membrane currents and contraction in uterine smooth muscle (rat)

1. The effects of prostaglandin E(1) have been studied on the transmembrane potentials, ionic currents, and contractions in isolated myometrial strips from pregnant rats by means of a double sucrose gap apparatus.2. At low concentrations (10(-8) g/ml.), prostaglandin E(1) reduced the duration (though not the amplitude) of the action potential, but significantly increased the contraction. The inward current was unchanged, as well as the phasic component of the contraction. However, the tonic contraction, recorded when the transmembrane calcium influx was blocked selectively with D 600, was stimulated significantly, and the outward current secondarily increased.3. At maximally effective doses (10(-6) g/ml.), the electrical response to prostaglandin E(1) consisted of a slight depolarization, while a large contracture developed. The depolarization and contracture were unaffected by the removal of external calcium. The inward current was reduced by prostaglandin E(1) and the reversal potential was shifted towards less positive values of voltage, indicating a decrease in the driving force and consequently, an increase in the internal calcium concentration.4. Dibutyryl-c-AMP (5 x 10(-4)M) produced a marked relaxation of the resting tension and a slight hyperpolarization of the uterine membrane. Under these conditions, a triggered action potential was able to evoke a larger contractile response. However, prostaglandin E(1) is known to increase tissue c-AMP, so that its contractile effect cannot be mediated by c-AMP.5. It is suggested that prostaglandin E(1) acts essentially by increasing the intracellular calcium concentration. This might result from the translocation of membrane-bound calcium from surface microvesicles or sarcoplasmic reticulum. The increase in internal calcium concentration could, in turn, lead to an increase in the outward current intensity.

The influence of calcium on sodium efflux in squid axons.

1. Previous work has shown that the sodium efflux from the axons of Loligo forbesi increases when external sodium is replaced by lithium.2. The increase in efflux in lithium was unaffected by ouabain but was abolished by removal of external calcium; in these respects it differed from the potassium-dependent sodium efflux which was abolished by ouabain but not reduced by removal of external calcium.3. Strontium but not magnesium could replace calcium in activating the ouabain-insensitive sodium efflux; lanthanum had an inhibitory effect.4. Replacing all the external NaCl by choline chloride or dextrose gave a rise in Na efflux which was abolished by ouabain but not by removal of external calcium.5. The rise in Na efflux resulting from partial replacement of NaCl by dextrose or choline chloride consisted of two components one of which was ouabain-insensitive and calcium-dependent and the other was inhibited by ouabain but calcium-insensitive.6. The ouabain-insensitive component of the Na efflux was activated by low concentrations of Na, Li or K but inhibited by high concentrations of Na and to a lesser extent Li. The inhibiting effect of high Na was of the kind expected if these ions displace calcium from an external site.7. The ouabain-insensitive component of the Na efflux was abolished by cyanide, had a Q(10) of 2.7; and was roughly proportional to [Na](i) (2). It was much more variable in magnitude than the ouabain-sensitive, potassium-dependent component of the sodium efflux.8. The calcium influx increased five to fortyfold when external NaCl was replaced by LiCl or dextrose, the increase for Li being larger than the increase for dextrose.9. The calcium influx from Na, Li or dextrose sea water was increased three to tenfold by increasing the internal Na about fourfold.10. The experiments provide evidence for a coupling between an inward movement of calcium and an outward movement of sodium.