Calmodulin In Ca2 Research Articles

Effect of aluminium on the NAD+ kinase activity of Euglena gracilis grown heterotrophically

The effect of 2 mM AlCl3 on NAD+ kinase (E.C. 2.7.1.23) activity was studied using Euglena gracilis strain Z grown heterotrophically in darkness at pH 3.5 in the presence of lactate as sole carbon source. The Al-treatment slowed down the culture growth and suppressed the peak of NAD+ kinase activity, which characterizes the beginning of the exponential phase of growth of the control cell cultures. There are two possible explanations of the Al effect: it 1) either prevents the enzyme activation by the Ca-calmodulin (CaM) complex; or 2) suppresses the CaM-dependent NAD+ kinase form. In Euglena cells, a part of the NAD+ kinase activity is enhanced by EGTA and lowered by Ca2+: this peculiar NAD+ kinase activity is unaffected by the Al treatment.

Enzyme dynamic study on calmodulin Ca++-Mg++-ATPase system of red blood cells inhibited by α-anordrin and probimane

To clarify the possible mechanisms of α-anordin and probimane on calmodulin Ca++-Mg++ ATPase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentration (ATP)-response curve, dose (inhibitors)-response curve and time-response curve]. Our data have shown that the inhibitory rates of α-anordrin and probimane are unrelated to substrate (APT) concentrations, but related to calmodulin concentrations. The inhibition of α-anordrin and probimane is very quick that is completed within 1 to 5 min and can maintain more than 1 hr in the same inhibitory rates. So it is possible that α-anordrin and probimane are calmodulin competitors with calmodulin-like binding whose actions can occur by affecting the reaction balance of substrate and product on target enzymes of calmodulin (Ca++-Mg++-ATPase).

Calmodulin regulation of light adaptation and store-operated dark current in Drosophila photoreceptors.

Phototransduction in Drosophila occurs through inositol lipid signaling that results in Ca2+ mobilization. In this system, we investigate the hitherto unknown physiological roles of calmodulin (CaM) in light adaptation and in regulation of the inward current that is brought about by depletion of cellular Ca2+ stores. To see the effects of a decreased Ca-CaM content in photoreceptor cells, we used several methods. Transgenic Drosophila P[ninaCDeltaB] flies, which have CaM-deficient photoreceptors, were studied. The peptide inhibitor M5, which binds to Ca-CaM and prevents its action, was applied. A Ca2+-free medium, which prevents Ca2+ influx and thereby diminishes the generation of Ca-CaM, was used. The decrease in the Ca-CaM level caused the following effects. (i) Fluorescence of Ca2+ indicator revealed an enhanced light-induced Ca2+ release from internal stores. (ii) Measurements of the light-induced current in P[ninaCDeltaB] cells showed a reduced light adaptation. (iii) Internal dialysis of M5 initially enhanced excitation and subsequently disrupted the light-induced current. (iv) An inward dark current appeared after depletion of the Ca2+ stores with ryanodine and caffeine. Importantly, application of Ca-CaM into the photoreceptor cells prevented all of the above effects. We propose that negative feedback of Ca-CaM on Ca2+ release from ryanodine-sensitive stores mediates light adaptation, is essential for light excitation, and keeps the store-operated inward current under a tight control.

Some properties of spectrin-like proteins from Pisum sativum.

Proteins cross-reacting with antibodies directed against alpha- and beta-spectrin were recently detected in plant cells. In this report we have studied the ability of these proteins to interact with other components of membrane skeleton such as ankyrin, f-actin and calmodulin. It was found that the polypeptide of high molecular weight reacting with anti-alpha-spectrin antibody binds calmodulin in Ca(2+)-dependent manner. Protein complexes containing polypeptides cross-reacting with anti-spectrin antibodies interact with muscle f-actin (in co-sedimentation assay) and with erythrocyte ankyrin (ELISA-type assay). These data further substantiate a possibility of occurrence of spectrin-based membrane skeleton in higher plant cells.

Evidence for a calmodulin-dependent phospholipase A2 that inhibits Na-K-ATPase.

We tested whether calnaktin, a proposed Ca/calmodulin (CaM)-dependent protein that inhibits the Na-K-ATPase, was a kinase, a phosphatase, a phospholipase A2 (PLA2), or a Ca-dependent protease. Human red blood cell membranes were extracted to remove associated calmodulin but to retain the proposed endogenous calnaktin. Exclusively cytoplasmic proteins and cofactors were presumably absent. In these membranes, free Ca inhibited the Na-K-ATPase with an inhibition constant (K[i]) of > or = 9 microM at a Na concentration of 18 mM. Addition of 100 nM CaM decreased the Ki to < 2 microM and increased the percent inhibition at 2 microM free Ca from 18 +/- 1 to 68 +/- 2%. The inhibitory effect of Ca/CaM was reversible, indicating that calnaktin is not a protease. Neither staurosporine (500 nM), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpipera zin e (5 microM), nor genistein (100 microM) diminished Ca/CaM inhibition of the Na-K-ATPase. Thus there is no evidence that this protein is a kinase. Likewise, the phosphatase inhibitors microcystin (1 microM) and okadaic acid (10 microM) had no effect. PLA2 inhibitors arachidonyl trifluoromethyl ketone (AACOCF3), parabromophenacyl bromide (pBPB), and quinacrine all abolished Ca/CaM inhibition of the Na-K-ATPase. Ca/CaM also increased PLA2 activity, as reflected by an increase in the slope of fluorescence signal of 10-pyrene phosphatidylcholine, a substrate for PLA2. This Ca/CaM-induced change in slope was inhibited by both pBPB and AACOCF3. These data suggest that human red cell membranes contain a form of PLA2 that is activated by Ca/CaM and that this enzyme may mediate Ca/CaM inhibition of the Na-K-ATPase.

Involvement of calmodulin in Ca 2+-activated K + efflux in human colonic cell line, HT29-19A

The receptor-mediated agonist, neurotensin (NT) stimulated Ba 2+- and charybdotoxin-sensitive 86Rb (K +) efflux in the HT29-19A colonic cell line. Efflux was also stimulated by ionomycin and thapsigargin and could be abolished by incubation with the intracellular Ca 2+ chelator, BAPTA. Together, these data suggest a rise in [Ca 2+] i is prerequisite for activation of K + efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca 2+] i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca 2+] i was transient, returning to baseline within 4–5 min, while efflux was sustained over a much longer period (>12 min). Ca 2+-activated 86Rb efflux was inhibited by pretreatment with calmodulin (CaM) antagonist, W7. W7 had no effect on basal efflux, but reduced both NT- and IM-activated efflux up to 80%, with a K i of 38 μM. Other CaM antagonists inhibited efflux with an order of potency (TFP ≈ W8 > W7 ⪢ W5) consistent with inhibition of a CaM-dependent process. Inhibition by W7 was not abolished by ouabain or bumetanide, indicating its effects are not mediated by action upon K + uptake processes. W7 did not inhibit NT-stimulated 125I efflux but significantly reduced efflux stimulated by the Ca 2+ ionophore, ionomycin. NT-stimulated 86Rb + efflux was localized to the basolateral membrane of HT29-19A monolayers grown on permeable supports. These data are consistent with the involvement of CaM in mediating Ca 2+-dependent activation of K + conductance in HT29-19A colonocytes.

Role of Calcium/Calmodulin-Mediated Processes in Protozoa

Publisher Summary This chapter describes the roles of calcium and calmodulin (CaM) in various protozoan systems and examines the structure of Tetrahymena CaM, its localization and cytology, and the regulation of specific enzyme systems. The presence of CaM in protozoa was first reported in Tetrahymena. While the structure of CaM is conserved in nature, Tetrahymena CaM possesses several unique structural characteristics that account for the biochemical and functional properties peculiar to this ciliate. CaM is a multifunctional protein with a molecular mass of 16,680 containing 148 amino acids, a high proportion of which are acidic residues. Following the stimulation of a cell, there is an increase in the intracellular free Ca2+ concentration due to the movement of Ca2+ through channels in the plasma membrane or due to its release from intracellular organelles. Each CaM molecule can bind as many as four Ca2+. The binding of Ca2+ produces conformational changes to a more helical structure that exposes hydrophobic regions, which, in turn, can bind to and activate CaM-binding proteins. The CaM–Ca2+complex can function directly and rapidly on an effector system, such as the ATPase transport system, or indirectly and slowly on a regulatory system, such as a protein kinase.

Regulation of smooth muscle myosin light chain kinase: Allosteric effects and co-operative activation by calmodulin

The activation of smooth muscle myosin light chain kinase (MLCKase) by calcium and calmodulin (CM) was investigated over a wide range of concentrations of the enzyme using myosin (MY) or its isolated phosphorylatable light chain (L 20) as substrates. The enzyme showed allosteric behavior. The specific phosphorylation activity was dependent on the concentration of MLCKase as well as on the concentrations of both substrates. However, at the lower (nanomolar) range of kinase the corresponding substrate rate relationships were hyperbolic. A high positive level of co-operativity of kinase was also observed for activation by CM in the presence of Ca 2+. There was a pronounced CM Ca -dependent inhibition of MLCKase activity when its molar ratio to CM was four to one or more. These kinetic data suggested that MLCKase could exist in several oligomeric forms, with an inactive high molecular size form and an active low molecular size form (protomers and/or dimers). This conclusion was confirmed by gel filtration studies. CM was not directly involved in the oligomerization process but instead, the oligomeric kinase shared an increased affinity for CM.

Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C

Binding of trifluoperazine (TFP), a phenothiazine tranquilizer, to porcine brain calmodulin (CaM) and rabbit skeletal muscle troponin C (Tn C) was measured by an automated high-performance liquid chromatography binding assay using a molecular sieving column; 10 micrograms of either protein per injection is sufficient for determining TFP binding, and results are comparable to those obtained by equilibrium dialysis. Very little binding was observed to either protein in the absence of Ca2+ while in the presence of Ca2+ both proteins bind 4 equiv of TFP. Other characteristics of TFP binding however are different for each protein. For CaM, half-maximal binding occurs at 5.8 microM TFP, the Hill coefficient is 0.82, and the fit of the data to the Scatchard equation is consistent with four independent TFP-binding sites. Binding of one melittin displaces two TFP from CaM. Thus, there are two recognizable classes of TFP-binding sites: those that are displaced by melittin and those that are not. TFP causes an increase in the Ca2+ affinity of CaM, and three Ca2+ must be bound to CaM for TFP binding to occur. The studies also yielded a measure of the intrinsic affinity of three of CaM's Ca2(+)-binding sites that is in agreement with previous reports. For troponin C, half-maximal binding occurs at 16 microM TFP, the Hill coefficient is 1.7, and the data best fit the Adair equation for four binding sites. The measured constants K1, K2, K3, and K4 were 2.5 X 10(4), 6.6 X 10(3), 5.8 X 10(5), and 2.0 X 10(5) M-1, respectively, in 1 mM Ca2+ and were similar when Mg2+ was additionally included. TFP also increases troponin C's Ca2+ affinity, and it is the low-affinity, Ca2(+)-specific binding sites that are affected. These studies yielded a measure of the intrinsic affinity of these Ca2(+)-binding sites that is in agreement with previous measurements.

A fluorescence temperature-jump study on Ca2+-induced conformational changes in calmodulin

Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.

Glycation of calmodulin: chemistry and structural and functional consequences

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurohormonal regulation of calcium in the cell.

Neurohormone C (NC) is a glycopeptide isolated from bovine hypothalamus, which inhibits Ca-calmodulin (CaM)-dependent cAMP and cGMP phosphodiesterase (PDE) and is a regulator of Ca in the cell. Distribution of [45Ca]CaCl2 in the mitochondria and reticulum (SR) of heart and brain mitochondria and changes of Ca-binding proteins in these organelles under NC influence have been studied in the myocardium before and after isoproterenol-induced necrosis. Intraperitoneal administration of 80-100 mU of PDE inhibitory activity of NC to rats did not cause any noticeable changes in the protein content of intracellular organelles, but altered the affinity of certain proteins to 45Ca2+. This property of NC was especially noticeable after isoproterenol necrosis. Necrotic injury of the myocardium induced Ca2+ storage in the mitochondria and SR of brain, and decreased the Ca2+ concentration in myocardial mitochondria. NC injection to the animals with necrosis was followed by Ca2+ release from all the studied organelles.

CAMP‐mediated inhibitory effect of calmodulin antagonists on ciliary reversal of Paramecium

AbstractTo explore possible roles of calmodulin in Ca2+‐induced ciliary reversal, we tested the effects of calmodulin antagonists on Triton‐extracted models of Paramecium. In the extracted models prepared by the method of Naitoh and Kaneko [Science 176:523–524, 1972], the Ca2+ ‐induced ciliary reversal was not inhibited by calmodulin antagonists, trifluoperazine (TFP), or 5‐chloro‐l‐naphthalenesulphone amide (W‐7). However, in the presence of adenosine 3′,5′‐cyclic mono‐phosphate (cAMP), whose concentration is below the one that alters the ciliary direction, TFP inhibited ciliary reversal and the models swam forward at 10−5 M Ca2+. When the washing medium in the preparation of the extracted models was replaced with one containing MgCl2, the extracted model showed sensitivity to calmodulin antagonists without addition of cAMP; at 10−5 M Ca2+, 40 μM TFP or 100 μM W‐7 inhibited the ciliary reversal and the models swam forward. Such effect of antagonists was abolished by an inhibitor of cAMP‐dependent protein kinase. On the other hand, addition of cAMP enhanced the inhibitory effect of antagonists. These results suggest that calmodulin antagonists act to increase the extent of cAMP‐dependent protein phosphorylation that inhibits the Ca2+ ‐induced ciliary reversal.

Regulation of Contraction in Cardiac and Smooth Muscles

Newer aspects of the contractile and regulatory proteins of cardiac and smooth muscles are described. Contraction in both tissues depends on Ca2+. In heart muscle, a fast, direct Ca2+-trigger system involving the troponin complex is operative on the actin filaments. Both the Ca2+-binding troponin-C and the phosphorylatable troponin-I display evolutionary diversification of properties characteristic of the heart muscle. Further modulation of heart-muscle contraction is governed by phosphorylation reactions, which are dependent on either Ca-calmodulin (CM) or cAMP. Both these regulatory pathways are operative in parallel. In smooth muscle, no fast Ca2+-trigger system is present. Smooth-muscle contraction is regulated by two systems, which operate indirectly and slowly via covalent protein modification. One involves phosphorylation of the myosin regulatory light-chain, the other phosphorylation of caldesmon on the actin filaments. Both are dependent on the Ca2+-binding CM. The CM-dependent myosin light-chain kinase (MLCK) itself is, in smooth muscle only, subject to control by a cAMP-dependent phosphorylation system. Thus, drugs that cause an increase in cAMP in smooth muscle, unlike in heart muscle, may lead to relaxation without lowering cytosolic free Ca2+ concentration.

Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport

To study the role of calmodulin in regulation of rabbit ileal active electrolyte transport by Ca2+, the effects of the Ca2+-calmodulin antagonist naphthalenesulfonamides W12 and W13 and the weak Ca2+-calmodulin antagonist promethazine, a phenothiazine, were studied on basal ileal Na and Cl transport and on secretion stimulated by Ca2+ and by cAMP. The naphthalenesulfonamides and promethazine all stimulated basal ileal Na and Cl absorption. The stimulation of Na absorption was dependent on Cl in the bathing solutions, and the stimulation of Cl absorption was Na dependent. This suggests that the transport process stimulated was the neutral, linked NaCl absorptive process. W13, which is a better Ca2+-calmodulin antagonist but has similar hydrophobic properties to W12, stimulated active ileal absorption at a lower concentration than W12, suggesting that the naphthalenesulfonamide-induced stimulation of active ileal absorption was not due to the hydrophobic properties of these drugs but could be due to their effects on the calcium-binding protein calmodulin. W12, W13, and promethazine did not alter paracellular transport, not affecting dilution potentials or structural features of the paracellular pathway. W12 and W13 did not decrease the changes in active ileal Na and Cl transport caused by increasing ileal cAMP content or intracellular Ca2+. This suggests that calmodulin is not directly involved in the active electrolyte secretion caused by increased intestinal Ca2+ or cAMP.

Erythrocyte membrane Ca2+-pumping ATPase of hypertensive humans: reduced stimulation by calmodulin.

The Ca2+-pumping ATPase of erythrocyte plasma membranes of hypertensive humans (HTN) show, in the absence of calmodulin, a low Vmax comparable to that of the enzyme of the erythrocyte membranes of normotensive humans (NTN). Although the addition of calmodulin (1.5 micrograms per ml) increased the maximum activity of the calcium pump of membranes of HTN and NTN individuals by at least 2-fold and 4-fold, respectively, the activator protein partially purified from the erythrocytes of HTN individuals enhanced the activity of the enzyme in a fashion similar to that of the protein obtained from the haemolysate of NTN individuals. A determination of the dependence of the activity of the pump on concentration of ATP revealed that the Km (ATP) of the enzyme of membranes of HTN individuals is 52% higher than that of the enzyme of membranes of NTN individuals, while the Vmax (1.75 +/- 0.28 mumol ATP mg protein-1 h-1) of the pump is 46% lower in the membranes of HTN humans than that of the enzyme of membranes of normal individuals (3.25 +/- 0.42 mumol ATP mg protein-1 h-1). It seems likely from these results that elevated erythrocyte Ca2+ concentration associated with essential hypertension may be due to a defective interaction between the Ca2+-pumping ATPase and the calmodulin Ca2+ complex.

Studies on calmodulin-binding proteins (CaMBPs) in the cilia of Tetrahymena

As a first step to elucidate the involvement of calmodulin in Ca 2+-dependent regulation of ciliary motility, molecular species and properties of calmodulin-binding proteins (CaMBPs) in Tetrahymena cilia were investigated by a modified [ 125I]calmodulin overlay method. At least 36 kinds of CaMBPs were detected. All the CaMBPs bound to calmodulin in Ca 2+-dependent and calmodulin-specific manners, but they showed different Ca 2+-dependencies. Several of CaMBPs bound to calmodulin in the presence of 100 μM trifluoperazine, several did in the presence of 8 M urea, and a few of them were highly sensitive to trypsin digestion. Among these CaMBPs, we noticed a 95 000-dalton (D) CaMBP present in the outerdoublet microtubule fraction, which possessed some attributes of the calmodulin counterpart suggested from the results of our previous paper [12]. We discussed a possibility that this protein might correspond to one of the protein components Of the interdoublet link.

Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle-free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain-coated vesicles by extraction with 0.5 M TRIS-HCl followed by Sepharose CL-4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr approximately 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500-dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+-induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr approximately 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr approximately 50,000 and 16,500 bands. This treatment abolished the in vitro coat-forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr approximately 100,000 band. Disappearance of the Mr approximately 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr approximately 76,000 and 65,000. When the elastase-treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr approximately 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr approximately 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000-dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.

Quantitative aspects of the development of a hydrophobic binding site on calmodulin by calcium binding.

AbstractThe interactive binding by calmodulin of Ca2+ and 1‐anilinonaphthalene‐8‐sulfonate (1,8‐ANS) has been examined. In the presence of saturating levels of Ca2+, calmodulin develops one moderately strong binding site for 1,8‐ANS, plus one or more weaker sites. The binding of 1,8‐ANS by unliganded, or singly liganded, calmodulin is slight; the development of a strong binding site, as well as the characteristic fluorescence enhancement and CD spectrum, requires the binding of two Ca2+ ions. Little further change occurs on binding additional Ca2+ ions.

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C.

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.