The uptake of calcium by isolated chromaffin granules of the adrenal medulla

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca²⁺ sequestration pathways were characterized. The rate of Ca²⁺ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca²⁺/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min^–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca²⁺/mg protein sequestered in 60 min. Ca²⁺ sequestration was 93% inhibited by 180 mM NaCl [I_50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl₂. Ca ²⁺ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca ²⁺ sequestration was dependent on extravesicular Ca ²⁺ with half-maximal sequestration at pCa²⁺ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca²⁺ could be exchanged with external ⁴⁵Ca²⁺, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca²⁺/mg (mean ± S.D., n = 3). The Ca²⁺/Ca²⁺ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl₂. About 75% of sequestered ⁴⁵Ca²⁺ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered ⁴⁵Ca²⁺ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na⁺-induced release of sequestered ⁴⁵Ca²⁺ was rapid, t_0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with ⁴⁵Ca²⁺ and either annexin proteins V or VI resulted in attenuated uptake of ⁴⁵Ca²⁺. These results suggest that Ca²⁺ uptake in adrenal chromaffin granules is regulated by Na⁺ and Ca²⁺ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca²⁺ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl₂ - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl₂     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

PTH release stimulated by high extracellular potassium is associated with a decrease in cytosolic calcium in bovine parathyroid cells

We employed the calcium (Ca⁺⁺)-sensitive, intracellular dye QUIN-2 to examine the role of cytosolic Ca⁺⁺ in the stimulation of PTH release by high extracellular potassium (K⁺) concentrations. Addition of 55 mM KCl to cells incubated with 115 mM NaCl and 5 mM KCl lowered cytosolic Ca⁺⁺ at either low (0.5 mM) extracellular Ca⁺⁺ (from 194±14 to 159±9 nM, p<.01, N=6) or high (1.5 mM) extracellular calcium (from 465±38 to 293±20 nM, p<.01, N=10). This reduction in cytosolic Ca⁺⁺ was due to high K⁺$\text{per}\text{se}$ and not to changes in tonicity since addition of 55 mM NaCl was without effect while a similar decrease in cytosolic Ca⁺⁺ occurred when cells were resuspended in 60 mM NaCl and 60 mM KCl. PTH release was significantly (p<.01) greater at 0.5 and 1.5 mM Ca⁺⁺ in QUIN-2-loaded cells incubated with 60 mM NaCl and 60 mM KCl than in those exposed to 115 mM NaCl and 5 mM KCl. In contrast to most secretory cells, therefore, stimulation of PTH release by high K⁺ is associated with a decrease rather than an increase in cytosolic Ca⁺⁺.     

The binding of arsenazo III to cell components

The Ca²⁺ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1–2 mM arsenazo III, the estimated free arsenazo III concentration is only 50–200 μM; 80–90% of the bound arsenazo III is associated with soluble proteins.The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca²⁺; this is due to a decrease in the affinity of the protein-bound dye for Ca²⁺. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca²⁺, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye.The Ca²⁺ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.     

Depolarization-induced release of l-glutamic acid from isolated-resealed synaptic membrane vesicles

l-Glutamic acid actively loaded into resealed brain synaptic membrane vesicles was rapidly released into the incubation medium following the introduction of KCl and CaCl₂, or nigericin, or veratridine into the external medium. The KCl-induced release was enhanced by the presence of low (0.1 mM), extravesicular [Ca²⁺]. Neither the KCl-induced nor the veratridine-stimulated l-glutamate efflux were carrier-mediated processes. Finally, the KCl-stimulated l-glutamate efflux was dependent on the ratio of intra- to extravesicular [K⁺]. The observations described in this study were indicative of depolarization-induced l-glutamate release from isolated synaptic plasma membrane vesicles.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

Carbachol- and KCl-induced changes in phosphoinositide metabolism and free calcium in guinea pig cerebral cortex synaptosomes

Phosphoinositide (PI) and calcium metabolism were studied in guinea pig cerebral cortex synaptosomes. Mass amounts of inositol and inositol monophosphates, and the levels of free intrasynaptosomal calcium ([Ca²⁺]_i) were measured after KCl (60 mM), after a direct cholinergic agonist carbachol (CA, 1mM), and after their combination. Inositol, inositol-1-phosphate (Ins1P), inositol-4-phosphate (Ins4P) and [Ca²⁺]_i were measured with and without 10 mM LiCl in the incubation medium. CA-induced cholinergic stimulation elevated synaptosomal Ins4P levels by 40% but did not affect Ins1P or [Ca²⁺]_i. On the contrary, KCl elevated Ins1P by 50% and [Ca²⁺]_i by 40% above the resting level, and decreased inositol by 20%, whereas no alterations in Ins4P occurred. CA did not modify the response of KCl, but KCl abolished the elevation of Ins4P by CA. LiCl attenuated KCl-induced elevation of Ins1P but amplified the CA-induced elevation of Ins4P. The elevation of presynaptic [Ca²⁺]_i was accompanied by accumulation of Ins1P but not that of Ins4P. Hence, the present results suggest that presynaptic cholinergic stimulation and KCl-induced depolarization may activate different degradation pathways of inositolphosphate metabolism.     

K+-and Mg2+-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca²⁺ + Mg²⁺-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg²⁺ and EGTA and is stimulated by Ca²⁺. The Mg²⁺-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl₂, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca²⁺ causes no further increase in the rate of hydrolysis and Ca²⁺ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca²⁺ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by P_i unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect P_i inhibition of the Mg²⁺-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a P_i-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and P_i. In this conformation the enzyme is transformed from a Ca²⁺- and Mg²⁺-dependent ATPase into a (K⁺ + Mg²⁺)-ATPase.     

Ca2+ homeostasis,Ca2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca²⁺ homeostasis and provide for Ca²⁺ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca²⁺ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca²⁺ homeostatic molecules on cytosolic Ca²⁺ ([Ca²⁺]_i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca²⁺]_i dynamics. When bathing the cells in a Na⁺-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca²⁺-ATPase (PMCA), La³⁺, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca²⁺]_i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca²⁺ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca²⁺ homeostatic pathways, the Na⁺/Ca²⁺ exchanger, the endoplasmic reticulum Ca²⁺ pump, the plasmalemmal Ca²⁺ pump and mitochondria, are complementary in actively clearing Ca²⁺ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca²⁺]_i dynamics; (iii) there is (are) Ca²⁺ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca²⁺ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

The membrane ATPase of Escherichia coli. II. Release into solution,allotopic properties and reconstitution of membrane-bound ATPase

Membrane-bound ATPase of Escherichia coli was released in a soluble form by decreasing the Mg²⁺ concentration to 0.05 mM. The particulate fraction left behind was depleted by more than 90% from its initial ATPase activity.Soluble ATPase exhibits a number of different properties as compared with membrane-bound ATPase. These are a 2-fold increased K_m toward ATP, a shift of 1–1.5 pH units in the pH-dependence curve, a greatly increased resistance to inhibition by N,N′-dicyclohexylcarbodiimide (DCCD) and a stimulation by Dio 9 instead of an inhibition.Upon mixing the soluble fraction and the depleted membrane fraction, the initial properties of native membrane-bound ATPase reappear. This reconstitution requires Mg²⁺ and results in the physical binding of the activity to sedimentable material.Soluble ATPase and depleted membrane can be titrated against each other until an equivalence point is reached, beyond which the component in excess keeps its previous characteristics.During the release procedure, DCCD remains associated with the particulate fraction with conservation of the ATPase-binding sites.Such DCCD-treated depleted membranes behave as a specific inhibitor of soluble ATPase.     

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca2+ release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

Experiments were performed to characterize the properties of the intrinsic Ca²⁺ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca_T]_SR and [Ca²⁺]_SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca²⁺ indicator). Results indicate SR Ca²⁺ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca²⁺. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca²⁺]_SR and [Ca_T]_SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca²⁺ permeability of the SR, namely d[Ca_T]_SR/dt ÷ [Ca²⁺]_SR (denoted release permeability), in experiments in which only [Ca_T]_SR or [Ca²⁺]_SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca²⁺ release of 2.3 SR Ca²⁺ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [Ca_T]_SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca²⁺]_SR inhibits release when [Ca_T]_SR declines to a low level.     

Calcium-dependent adenylate cyclase of pituitary tumor cells

Effects of Ca²⁺ and calmodulin on the adenylate cyclase activity of a prolactin and growth hormone-producing pituitary tumor cell strain (GH₃) were examined. The adenylate cyclase activity of homogenates was stimulated approx. 60% by submicromolar free Ca²⁺ concentrations and inhibited by higher (μM range) concentrations of the cation. A 2–3-fold stimulation of the activity in response to Ca²⁺ was observed at physiologic concentrations of KCl, with both the stimulatory and inhibitory responses occurring at respectively higher free Ca²⁺ concentrations. Calmodulin in incubations at low KCl concentrations increased the enzyme activity at all Ca²⁺ concentrations tested. In incubations conducted at physiologic KCl concentrations, both the inhibitory and stimulatory responses to Ca²⁺ were shifted by calmodulin to lower respective concentrations of the cation, without significant change occurring in the maximal rate of enzymic activity at optimal free Ca²⁺. Mg²⁺ concentrations in the incubation also influenced the Ca²⁺ concentration dependence of adenylate cyclase; at high Mg²⁺ more Ca²⁺ was required to obtain maximal activity. Trifluoperazine inhibited adenylate cyclase of GH₃ cells only in the presence of Ca²⁺; as Ca²⁺ concentrations in the assay were increased, higher drug concentrations were required to inhibit the enzyme. Ca²⁺ was also observed to reduce the extent of enzyme destabilization which occurred during pretreatments at warm temperatures. Vasoactive intestinal polypeptide and phorbol myristate acetate, which stimulate prolactin secretion in intact GH₃ cells, enhanced enzyme activity 4- and 2.5-fold, respectively, without added Ca²⁺. Increasing free Ca²⁺ concentrations reduced the enhancement by VIP and eliminated the stimulation by PMA.     

Release of Sucrose from Vicia faba L. Leaf Discs

The release of sucrose from leaf discs of Vicia faba L. to a bathing medium was studied for evidence of a relationship between this release and mesophyll export of photosynthate in vivo. Sucrose was released specifically over hexoses and represented over 85% of total photosynthate released. The sucrose appeared to be derived from the mesophyll tissue directly and release did not require concurrent photosynthesis. The data indicated two separate channels for sucrose release. The first was sensitive to inhibition by 1 millimolar p-chloromercuribenzenesulfonic acid and the second was promoted by lowering the Ca²⁺ concentration below 0.1 millimolar. Flow through both channels was about equal when tissue that had been actively photosynthesizing for several hours was used. The rate of release was not dependent on the extracellular pH, but was inhibited by 10 micromolar carbonylcyanide p-trifluromethoxyphenylhydrazone. Lowering the Ca²⁺ concentration below 0.1 millimolar or raising the K⁺ concentration above 100 millimolar stimulated sucrose release. The stimulation by high K⁺ was not reversed by adding Ca²⁺. The data supported the postulate that Ca²⁺ removal or K⁺ addition changed the permeability of the mesophyll plasma membrane to sucrose.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

LHRH-receptor-regulated Ca2+-ATPase activity in murine pituitary gland

Addition of luteinizing hormone releasing hormone (LHRH) in vitro (10^–5–5×10^–9 M) to murine pituitary membranes resulted in a dose-related decrease in Ca²⁺-ATPase activity within 15 min. Inhibitory effects of LHRH (10^–7 M) occurred after 90 sec, and appeared maximal by 120 sec. Eadie-Hofstee analysis at 10^–7 M LHRH, at varying [Ca²⁺]_free, resulted in aK _m=0.89±0.06 M and aV _max=18.8±0.71 nmol/mg per 2 min, compared to aK _m=0.69±0.06 M and aV _max=32.8±1.21 nmol/mg per 2 min for controls. Pre-incubation for 5 min with LHRH antagonist (10^–8 M) significantly attenuated (50%) the inhibitory effects of 10^–7 M LHRH on pituitary Ca²⁺ ATPase activity with aK _m=0.97±0.24 M and aV _max=28.1±2.8 nmol/mg per 2 min. The addition of LHRH (10^–7 M) to pituitary homogenates significantly increased luteinizing hormone (LH) release already at 10 and up to 40 sec compared to basal LH release. Systemic administration of 50 ng LHRH (i.p.), significantly (P<0.05) reduced pituitary Ca²⁺-ATPase after 30, 60 and 90 min, with a return to control levels by 120 min. Pituitary LH content was reduced slightly at 15 min, but was increased significantly at 90 and 120 min post-treatment. Plasma LH levels were elevated by 5 min, reached a peak by 15 min and returned to control within 60 min. The present findings indicate that LHRH receptor activation may influence cytosolic Ca²⁺ transport through effects on membrane Ca²⁺-ATPase activity. These actions may regulate LHRH-induced synthesis, storage and release of LH from pituitary gonadotropes.     

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca²⁺ ions in the cytoplasm ([Ca²⁺] _i ) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca²⁺] _i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca²⁺] _i was observed in all tested neurons. To study the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.     

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

To investigate the presence and the size of different non-mitochondria) Ca²⁺ pools of Ehrlich ascites tumor cells (EATCs) , digitonin-permeabilized cells were allowed to accumulate Ca²⁺ in the presence of mitochondrial inhibitors and treated with the reticular Ca²⁺-ATPase inhibitor thapsigargin, IP₃ and the Ca²⁺ ionophore A23187. Emptying of thapsigargin-sensitive Ca²⁺ stores prevented any Ca²⁺ release by IP₃, and, after IP₃ addition, little or no Ca²⁺ was released by thapsigargin. In both instances, a further Ca²⁺ release was accomplished by A23187. The IP₃-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondria) stored Ca²⁺, respectively. In intact EATCs, IP₃-dependent agonists and thapsigargin discharged Ca ²⁺ pools almost completely overlapping, and A32187 released a minor residual Ca²⁺ pool. The IP₃-insensitive pool appeared to have a relatively low affinity for Ca²⁺ (below 600 nM). The high affinity, IP₃-sensitive Ca²⁺ pool was discharged in a ‘quantal’ manner following step additions of sub maximal [IP₃], and the IP₃-induced fractional Ca²⁺ release was more marked at higher concentrations of stored (luminal) Ca²⁺, The IP₃-sensitive Ca²⁺ pool appeared to be devoid of the Ca²⁺-activated Ca²⁺ release channel since caffeine did not released any Ca²⁺ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.