Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.     

Stimulatory effect of calcitonin on calcium uptake and glucose production in isolated rat hepatocytes

The biological effect of calcitonin (CT) was investigated in isolated rat hepatocytes. Addition of synthetic [Asu1,7] eel CT (20 and 40 ng/ml of medium with 2.5 mg dry weight of cells) into the medium containing calcium ion produced a marked elevation of calcium uptake in the hepatocytes, when calcium concentrations in the medium were monitored spectrophotometrically using arsenazo III. Intracellular calcium contents in the mitochondria and microsomes were significantly increased by CT addition. Meanwhile, glycogenolysis and gluconeogenesis in the hepatocytes were significantly stimulated by addition of CT (10, 10(2), and 10(3) ng/ml of medium with 5 mg dry weight of cells) into the medium containing calcium, although CT effects were less than those effects of 10(-7) M glucagon and 10(-5) M phenylephrine. These data clearly indicate that CT stimulates calcium uptake and glucose production in the hepatocytes, suggesting that CT has an effect in the metabolic function of rat liver cells.     

The effect of local anesthetics on calcium transport across the rat and turtle small intestine

Procaine at different concentrations enhanced significantly (P less than 0.01) calcium accumulation in rat intestinal cells, whereas the same concentrations of procaine inhibited significantly (P less than 0.01) calcium uptake by the turtle small intestine. Unidirectional calcium influx across the rat small intestine was significantly enhanced (P less than 0.001) by the presence of procaine in the preincubation medium. However, procaine had no effect on calcium influx across the turtle intestinal cells. The cell water content and the cell volume were not altered by preincubating the intestinal tissues with procaine in both animals.     

Enhanced uptake of calcium by transforming lymphocytes

Phytohemagglutinin caused a rapid increase in calcium accumulation by lymphocytes. The enhanced uptake was observed within 1 hr of initiation of transformation in both human lymphocyte and mouse spleen cell cultures. Increased uptake was also found in mixed lymphocyte cultures although not until late in the response. The rate of calcium uptake increased with time after stimulation and depended upon the PHA concentration. The lowtemperature coefficient (Q₁₀) for calcium permeability in unstimulated cells was indicative of a passive diffusion process, but the Q₁₀ was slightly greater for PHA-stimulated cells. Various chemical agents which alter membrane properties and/or cellular metabolism inhibited uptake to a greater extent in stimulated cultures than in control cultures. Ouabain did not affect the calcium permeability of controls or stimulated cells within 1 hr after PHA addition, but it partially inhibited calcium uptake 12 hr after PHA treatment. Cyclic AMP, dibutyryl cyclic AMP, and theophylline also altered calcium transport providing evidence for an effect of cyclic AMP on an early event in the transformation process.     

Transverse tubule calcium regulation in malignant hyperthermia

Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca2+ concentration (K1/2 for Ca2+ of 0.21 +/- 0.04 and 0.25 +/- 0.05 microM for MHS and normal TT, respectively), as well as a similar Vmax (20.9 +/- 2.0 and 23.7 +/- 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or cAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 mM inhibited calcium uptake by either normal or MHS TT to a similar extent (IC50 = 8 mM). Dantrolene (10 microM), nitrendipine (1 microM), and Bay K 8644 (1 microM) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 +/- 10 versus 130 +/- 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH crisis.     

N-Methyl-D-Aspartate Receptors and Ethanol: Inhibition of Calcium Flux and Cyclic GMP Production

Measurements of calcium uptake and cyclic GMP production by cerebellar granule cells grown in primary culture demonstrated that ethanol preferentially inhibited N-methyl-D-aspartate (NMDA) receptor-gated cation channel function. Concentrations of ethanol as low as 10 mM inhibited NMDA-stimulated Ca2+ uptake by greater than 30%, and ethanol also inhibited NMDA-stimulated (Ca2+-dependent) cyclic GMP accumulation in a similar, dose-dependent manner. Responses to kainate were significantly less sensitive to ethanol. Studies using various concentrations of NMDA, as well as phencyclidine (PCP) and glycine, suggested that ethanol affected the "coagonist" binding site of the NMDA receptor-channel complex, rather than the PCP recognition site.     

Seasonal variations in the rate and capacity of cardiac SR calcium accumulation in a hibernating species.

The rate of calcium uptake and the level of calcium accumulation was measured in cardiac muscle SR from hibernating and nonhibernating Richardson's ground squirrels. In whole heart homogenates, the rate of calcium uptake was higher (P less than 0.05) in hibernating animals than it was in active animals. Further purification of homogenates into sacroplasmic reticulum (SR) preparations showed that the hibernating animals had the highest rate of calcium uptake and the greatest level of calcium accumulation. These results could not be explained by variations in non-SR membrane contaminants nor by changes in the maximal activity or total amount of a SR marker enzyme, the Ca(2+)-ATPase. The addition of ryanodine to the calcium uptake medium increased the level of calcium accumulation in all groups by a similar amount. It is concluded that the high rate of calcium uptake by isolated cardiac SR vesicles from hibernating ground squirrels reflects the activity of the organelle in vivo, and that the ability of the ryanodine-insensitive population of SR vesicles to accumulate calcium is affected by hibernation.     

Mechanism of Neurotransmitter Release Elicited by the Preferential α1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.     

Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte

Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium.     

Uptake and release of 45Ca by brain microsomes, synaptosomes and synaptic vesicles

Abstract— Microsomes and synaptosomes from rat brain accumulated ^4,5Ca against a concentration gradient by an ATP-dependent process. Calcium accumulation occurred to the same extent in microsomes prepared from white matter and from grey matter, an observation suggesting that calcium uptake may be in part an activity of the axonal membrane. Microsomes and synaptosomes accumulated calcium to a similar extent but less actively than mitochondria. By contrast, synaptic vesicles showed relatively little calcium accumulation. Isotonic concentrations of sucrose, NaCl, KCl and choline chloride inhibited calcium accumulation, with NaCl and KCl the least effective of these inhibitory agents. No consistent effects on calcium uptake were obtained with adenosine 3′,5′-monophosphate, dibutyryl cyclic AMP or the methyl xanthines. Incubation of prelabelled microsomes resulted in a release of ⁴⁵Ca, and ATP inhibited this release process. In the absence of added ATP, isotonic NaCl promoted calcium release to a significantly greater extent than KCl choline chloride or sucrose. In the presence of ATP, these agents all promoted a similar degree of release. Adenosine 3′,5′-monophosphate or agents that affect its metabolism did not significantly affect calcium release. Magnesium ions reduced calcium release under all conditions tested.     

Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of calcium ion in epinephrine activation of lipolysis.

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.     

Chylomicron-remnant uptake by freshly isolated hepatocytes. Effect of heparin and of hepatic triacylglycerol lipase.

Chylomicron remnants labelled biologically with [3H]cholesterol were efficiently taken up by freshly isolated hepatocytes during a 3 h incubation in Krebs bicarbonate medium. Their [3H]cholesteryl ester was hydrolysed (74% net hydrolysis), and 0.1 mM-chloroquine could partially inhibit this hydrolysis, provided that hepatocytes were first preincubated for 2 h 30 min at 37 degrees C. This hydrolysis was also measured in preincubated cells with remnants double-labelled (3H and 14C) on their free cholesterol moiety; [3H]cholesterol arising from [3H]cholesteryl ester hydrolysis was recovered in the free [3H]cholesterol pool. A dose-response study showed saturation of remnant uptake at 180 micrograms of remnant protein/10(7) cells. Heparin (10 units/ml) increased remnant uptake by 63% (P less than 0.01), [3H]cholesteryl ester accumulation in the cell pellet by 110% (P less than 0.025) and hepatic lipase activity secreted in the medium by 2.4-fold (P less than 0.01) and by 3.3-fold (P less than 0.01) at the end of the preincubation and incubation periods respectively. Addition of 100 munits of semi-purified hepatic lipase preparation/flask stimulated remnant uptake by 44-69%, and [3H]cholesteryl ester accumulation in the presence of chloroquine by 2.1-fold (P less than 0.025). When hepatic lipase was incubated solely with the remnants, it decreased their triacylglycerol and phospholipid contents by 24% and 26% respectively. Thus freshly isolated hepatocytes may be used to study chylomicron-remnant uptake. Hepatic lipase, which seems to underly the stimulating effect of heparin, facilitates remnant uptake in vitro, and this could be mediated by at least one (or both) of its hydrolytic properties.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

The effect of theophylline and dibutyryl cyclic AMP on the uptake of radioactive calcium and phosphate ions by boar and human spermatozoa.

Radioactive calcium uptake by suspensions of washed boar and human spermatozoa was inhibited by the mitochondrial uncoupling agent carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Theophylline + dibutyryl cyclic AMP also inhibited calcium uptake in the presence or absence of FCCP. Uptake of low concentrations of calcium (0 . 1 mM) was inhibited by the calcium ionophore A23187, but at high calcium concentrations the ionophore stimulated calcium uptake. These observations are explained in terms of a mechanism for the regulation of calcium uptake in spermatozoa based on competing mitochondrial and plasma membrane pumps. Uptake of 32P was also inhibited. These effects provide evidence that cyclic AMP plays a role in the transport of ions across the plasma membrane of spermatozoa.     

Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

Calcium uptake by subcellular fractions of pancreatic islets. Effects of nucleotides and theophylline.

Uptake of 45Ca2+ by a microsomal fraction isolated pancreatic islets of non-inbred ob/ob mice was studied. ATP strongly stimulated 45Ca2+ uptake, the maximum effect being obtained with 2mM-ATP. GTP and CTP at this concentration did not increase the uptake. Scatchard analysis revealed at least two types of uptake mechanisms in the presence of 2mM-ATP; the apparent association constants were 1.1 x 10(5)m(-1) and less than 2.5 x 10(2)m(-1). In contradistinction to an unaffected low-affinity uptake, the high-affinity uptake was drastically decreased on ommission of ATP. The ATP-dependent and high-affinity uptake was half-saturated at about 10-20mum-Ca(2+) and was inhibited by 10 or 100mum cyclic AMP, 10mum cyclic GMP, 10 mum cyclic GMP, or 5mm-theophylline. 45ca2+ uptake in the absence of ATP was not affected by 100mum-cyclic AMP. In view of its sensitivity to ATP and cyclic nucleotides, the high-affinity Ca2+-uptake mechaniam may play a role in stimulus-secretion coupling in the beta-cells by regulating the cytosolic concentration of Ca2+.     

Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle.

The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Evidence for a halothane-dependent cyclic flux of calcium in rat-liver mitochondria.

The previously reported (Hall et al., Biochem. Soc. Trans. 1973) halothane-dependent, calcium-induced loss of respiratory control in rat liver mitochondria is relatively specific to calcium; the effect of strontium ions is much smaller, and comparable additions of potassium salts have no effect on mitochondrial respiration on succinate in the presence of halothane. The calcium-dependent loss of respiratory control can be prevented, or reversed, respectively, by the prior or subsequent addition of agents that either chelate extramitochondrial Ca2plus or inhibit calcium accumulation, or that inhibit the efflux of accumulatec calcium. These results suggest that the halothane-dependent, calcijm-induced loss of respiratory control is due to a cyclic flux of calcium uptake and release.     

Effect of taurine on ATP-dependent calcium transport in guinea-pig cardiac muscle

The effect of taurine on ATP-dependent calcium transport was examined in guinea-pig cardiac ventricle homogenates and in microsomal preparations enriched in sarcoplasmic reticulum. Taurine (5?50 mM) did not affect ATP-dependent calcium binding or uptake in either of these preparations or alter the rate of decay of calcium uptake activity. Taurine (20 mM) also did not affect the oxalate-dependent calcium uptake stimulation noted in the presence of cyclic AMP-dependent protein kinase and cyclic AMP. The mechanism by which taurine alters cardiac function remains to be elucidated.