Calmodulin and calmodulin-mediated processes in plants

Abstract. The Ca²⁺ -binding protein calmodulin is found in all plants investigated so far. The comparison of the biochemical and functional properties reveals that it is structurally conserved and functionally preserved throughout the plant and animal kingdom. Among the plant enzymes so far known to be dependent on the Ca²⁺ -calmodulin complex are NAD kinase(s), Ca²⁺ -transport ATPase, quinate: NAD⁺ oxidoreductase, soluble and membrane bound protein kinases, and H⁺ -transport ATPase. Calmodulin may play also an important role in the regulation of other cellular reactions, such as hormone-mediated processes, secretion of enzymes, and contractile mechanisms. On the basis of the NAD kinase and its regulation by light and Ca²⁺ -calmodulin, it is suggested that changes in the cellular, free Ca²⁺ concentration following stimulation may alter the metabolism of a plant cell. According to this suggestion free Ca²⁺ may act as a second messenger in plants much as it does in animal cells.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Several compartments of Saccharomyces cerevisiae are equipped with Ca2+-ATPase(s)

Abstract Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of ⁴⁵Ca²⁺ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of ⁴⁵Ca²⁺ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca²⁺-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca²⁺-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.     

Induced Ca2+ uptake and callose synthesis in suspension-cultured cells of Catharanthus roseus are decreased by the protein phosphatase inhibitor okadaic acid

Subtoxic concentrations of the saponin digitonin. the polyene antibiotic amphotericin B and the bacterial phytotoxin syringomycin induce increased uptake of ⁴⁵Ca²⁺ into suspension-cultured plant cells and a rapid Ca²⁺-dependent defense response, callose synthesis. Both reactions were inhibited by preincubation of the cells with okadaic acid, a specific inhibitor of type 1 and type 2A protein phosphatases. These results suggest that Ca²⁺ uptake induced by the above agents does not occur due to unspecific perturbation of plasma membrane permeability but involves transport proteins which are controlled by protein phosphorylation/dephosphorylation. Phosphoproteins appear also to be involved in the regulation of callose synthesis, although it remains open whether this control is effected at the level of Ca²⁺ transport or at the 1,3-ß-glucan synthase involved in deposition of the polymer.     

Temperature dependence of the barley root plasma membrane-bound Ca2+- and Mg2+-dependent ATPase

Arrhenius plots of the maximal velocities for the Ca²⁺-and Mg²⁺-dependent ATPase activities found in a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) were nonlinear. Arrhenius plot analyses using a relation which produced curvilinear Arrhenius plots accurately fit the data and allowed the calculation of the activation enthalpies and molar heat capacities of activation. The temperature dependence of the computed Km values for the Ca²⁺- and Mg²⁺-dependent ATPase activities was complex, with the highest enzyme-substrate affinities being obtained near the barley seedling growth temperature (16°C). Using electron paramagnetic resonance spectroscopy with amphiphilic cationic and anionic spin probes, it was possible to demonstrate that temperature changes and increasing Ca²⁺ concentrations could alter the mobility of the membrane lipid polar head groups. Inhibition of the ATPase activities by high levels of Ca²⁺ may result from a Ca2+-induced reduction in the lipid polar head group mobility. The possible role of lipid polar head group-protein interactions in the complex temperature dependence of the barley root ATPase kinetic constants is discussed.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.     

ATPase activity associated with vacuoles and tonoplast vesicles isolated from the CAM plant, Kalanchoë daigremontiana

Intact vacuoles were isolated from leaves of the CAM plant, Kalanchoë daigremontiana Hamet et Perr. Both ATPase and acid phosphatase activities were found in the vacuoles. Purified tonoplast vesicles showed only ATPase activity with a pH optimum of 8.0. This activity was Mg²⁺-dependent and KCI or NaCI caused a further stimulation. N,N'-dicyclohexylcarbodiimide, diethylstilbestrol and quercetin inhibited the ATPase almost completely at concentrations well below 1 m M. NaVo₃, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, oligomycin and NaN₃ had little or no effect. Carbonyl cyanide m -chlorophenylhydrazone stimulated the ATPase about 40% at 5 × 10⁻⁴ M. The K_m for ATP was found to be 0.55 m M. These results indicate that the ATPase found in the tonoplast membrane of Kalanchoë daigremontiana is qualitatively similar to that of other plant species.     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Ca2+-dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat ( Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [³²P]-ATP into phosphoproteins was maximal in the presence of 1 m M CaCl₂ and 5 m M MgCl₂Ca²⁺ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin-layer electrophoresis showed that the Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca²⁺ dependent protein phosphorylktion was inhibited by phenothiazine-derived drugs. Addition of S-adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca²⁺-dependent protein phosphorylation.     

From common signalling components to cell specific responses: insights from the cereal aleurone

Studies into the molecules underlying plant signal transduction events continue to reveal the involvement of highly conserved factors such as Ca²⁺, calmodulin, cyclic GMP and phospholipases in a remarkably diverse array of physiological processes. The hormonal response systems in the aleurone cells of the cereal grain and in the stomatal guard cell are beginning to reveal how diversity of response can be hard wired into these cells despite the use of these common signalling intermediates. In both the aleurone and the guard cell ABA signalling operates through the action of phospholipase D and alterations in a Ca²⁺-dependent signalling system. The role of phospholipase D is highly analogous in these two divergent cell types, perhaps reflecting the closeness of this enzyme to a conserved ABA receptor. However, specificity in response becomes evident in elements downstream from PLD, such as in the Ca²⁺ signalling system. For example, ABA has opposite effects on cytoplasmic Ca²⁺ in the aleurone and guard cell. Combining the Ca²⁺-dependent signalling activities in networks with parallel regulatory activities such as cyclic GMP appears to underlie the flexible regulatory systems that are the hallmark of plant cell function.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membrane after treatment of intact tissues and cell free extracts with triacontanol

Ca²⁺- and Mg²⁺-dependent ATPase activity (EC 3.6.1.3) in a plasma membrane-enriched fraction increased rapidly after in vivo application of physiologically active concentrations of triacontanol (TRIA) to the roots of barley ( Hordeum vulgare L. cv. Conquest) seedlings. Ca²⁺- and Mg²⁺-dependent ATPase activity was 64 and 85% higher, respectively, in the roots of seedlings germinated in the presence of growth-promoting concentrations of TRIA compared to controls. The increase in vivo was concentration dependent, with the greatest increase obtained at 2.3 n M TRIA. Maximal stimulation of ATPase activity of excised tissue treated with TRIA coincided with the temperature at which the barley was grown. At this temperature the plasma membrane is primarily in a mixed gel/liquid crystalline state. Pretreatment of barley roots with cyclohexamide did not alter ATPase stimulation by TRIA. Two to three times more [¹⁴C]-TRIA (mg membrane protein)⁻¹ was found associated with plasma membrane-enriched vesicles treated with TRIA than with vesicles enriched for mitochondrial membranes or for vesicles enriched for tonoplast, Golgi and rough endoplasmic reticulum. Both Ca²⁺- and Mg²⁺-dependent ATPase activity increased by 40–60% within 30 min of the addition of 2.3 n M TRIA to cell-free extracts of barley roots. The addition of octacosanol, the C₂₈ analogue of TRIA, to cell-free extracts did not affect metal-dependent ATPase activity. Consistent with many studies in the green-house, simultaneous additions of equimolar amounts of TRIA and octacosanol to cell-free extracts resulted in inhibition of ATPase stimulation by TRIA. TRIA may directly affect plasma membrane function in barley roots.     

Picomolar concentrations of tentoxin affect Mg2+- and Ca2+-ATPase activities of plasma membrane vesicles from roots of winter wheat seedlings

Purified plasmalemma vesicles were isolated in the presence of 250 m M sucrose from roots of 14-day-old seedlings of winter wheat ( Triticum aestivum L. Martonvásári-8) by phase partitioning of salt-washed microsomal fractions in a Dextran-polyethylene glycol two-phase system, and both Mg²⁺- and Ca²⁺-ATPase activities were detected. Orthovanadate-sensitive Mg²⁺-ATPase activity associated with the inside of right side-out plasmalemma (PM) vesicles (latency 98%) was inhibited 76% by 0.3 m M Ca²⁺, Ca²⁺-dependent ATPase activity located partly on the inside and partly on the outside of plasmalemma vesicles (latency 47%) was not affected by Mg²⁺.
Mg²⁺-ATPase activity was inhibited by 68% and inhibition of Mg²⁺ activation by 0.3 m M Ca²⁺ partly disappeared in the presence of 10 p M tentoxin, a fungal phytotoxin. Mg²⁺-ATPase activity remained inhibited up to 10 n M tentoxin while at 1 μ M tentoxin Mg²⁺ activation was as high as without tentoxin. K⁺-stimulation and vanadate inhibition was increased and decreased, respectively, by 100 p M -10 n M tentoxin. Ca²⁺-dependent ATPase activity was continuously increased by 1 p M -10 n M tentoxin, but at 1 μ M tentoxin the stimulation disappeared. The effects of p M tentoxin on plasma-lemma Mg²⁺-ATPase are discussed in relation to its influence on K⁺ transport in wheat seedlings.     

ATPASE AND PHOSPHATIDYLINOSITOL KINASE ACTIVITIES OF ADRENAL CHROMAFFIN VESICLES

Abstract— The hypothesis that the ATPase and phosphatidyhnositol (PI) kinase activities of chromaffin vesicle membranes are catalysed by same enzyme was investigated. The two activities exhibited entirely different responses to variations in Mg²⁺ or Mn²⁺ concentrations. In the presence of 1 mM ATP, maximal ATPase activity occurred with 1 mM Mg²⁺ while maximal PI kinase activity required 100 mM Mg²⁺ Similar differences were observed with Mn²⁺ with the exception that maximal ATPase activity occurred with 0.5 mM Mn²⁺ and maximal PI kinase activity occurred with 5 mM Mn²⁺ Mn²⁺ was more effective than Mg²⁺ in stimulating PI kinase activity at low concentrations, but at optimal concentrations of each, the maximal activity obtained with Mg²⁺ was 5-fold greater than the maximal activity obtained with Mn²⁺ The heat stabilities of the two enzymes are vastly different. At 50°C the ATPase activity of the intact membranes was stable for up to 20 min while the t _l/2 of PI kinase was less than 2 min. After solubilization in Lubrol PX or at higher temperatures both enzymes were less heat stable, but PI kinase was still inactivated at a much greater rate than the ATPase. The evidence suggests that the ATPase and the PI kinase are different proteins.
The major phosphorylated product was diphosphatidylinositol and once formed, it was stable. Phosphorylation of membrane protein accounted for less than 10% of the total ³²P-incorporated into chromaffin vesicles. SDS gel electrophoresis of the solubilized membranes showed the presence of at least 2 major phosphorylated high molecular weight components.     

Neuronal Localization of Ca2+-dependent Protein Phosphorylation in Brain

Abstract: The cellular localization of two Ca²⁺-dependent protein phosphorylation systems was investigated using the kainic acid lesioning technique for the selective destruction of neurons. In one of these systems, a crude synaptosomal (P₂) fraction was preincubated with ³²P_j for 30 min; the phosphorylation of several proteins was increased during a short subsequent incubation with veratridine plus Ca²⁺. In the second system, crude synaptosomal membranes isolated from the P₂ fraction were incubated with [γ-³²P]ATP; in this system, the phosphorylation of several proteins was increased in the presence of a "calcium-dependent regulator" plus Ca²⁺. Kainic acid lesioning greatly reduced the amount of Ca-⁺-dependent protein phosphorylation in both systems. The results indicate a predominantly neuronal localization for both Ca²⁺-dependent protein phosphorylation systems.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Nitrate reductase in higher plants: A case study for transduction of environmental stimuli into control of catalytic activity

In higher plants, cytosolic NAD(P)H-nitrate reductase (NR) is rapidly modulated by environmental conditions such as light, CO₂, or oxygen availability. In leaves, NR is activated by photosynthesis, reaching an activation state of 60–80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity. In roots, hypoxia or anoxia activate NR, whereas high oxygen supply inactivates NR. Spinach leaf NR is inactivated by phosphorylation of serine 543 and subsequent Mg²⁺-dependent binding of 14-3-3 proteins at, or close to, this phosphorylation site. At least three different protein kinases (NR-PK) have been identified in spinach leaves that are able to phosphorylate NR on serine 543. Two of them show up as calmodulin-like domain protein kinases (CDPKs), and one as a SNF1-like protein kinase. Dephosphorylation of serine 543 is catalyzed by a Mg²⁺-dependent protein phosphatase and by a type 2A protein phosphatase (NR-PP), which is regulated by a trimer/dimer interconversion. The NR-PKs, NR-PPs, and 14-3-3s are present even in NR-depleted plant tissues. Artificial activation of NR in vivo is achieved by cellular acidification, by respiratory inhibitors, or by mannose feeding. As for anoxia, these treatments seem to act, at least in part, via cytosolic acidification, mediated by low cytosolic ATP levels. Activation is also achieved by ionophore-induced release of divalent cations from the cytosol. In addition, cytosolic AMP and phosphate esters seem to regulate NR-PK and NR-PP activities, thereby adapting NR activity within minutes to the changing environment.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.