The Persistent Membrane Retention of Desipramine Causes Lasting Inhibition of Norepinephrine Transporter Function

The present study examined the potential membrane retention of desipramine (DMI) following exposures of 293-hNET cells to DMI, and its effect on [3H]NE uptake. Incubation of cells with 500 nM DMI for 1 h or 1 day persistently inhibited the uptake of [3H]NE up to 7 days, despite daily repeated washing of cells with drug-free medium. Uptake inhibition was paralleled by persistent retention of DMI associated with cells, as determined by HPLC and by radiotracer experiments using [3H]DMI. [3H]DMI trapped in membranes was displaceable by the structurally unrelated NET inhibitor, nisoxetine, in a concentration-dependent manner, implying interaction of retained [3H]DMI with the NET. A similar cellular retention was observed following incubation of cells with nisoxetine. The results demonstrate that DMI and nisoxetine are persistently retained in cell membranes, at least partly in association with the NET. The retention and slow diffusion of DMI and nisoxetine from membranes may contribute to their pharmacological and modulatory action on NET.     

Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus

We have previously reported that atrial natriuretic factor (ANF) increased neuronal norepinephrine (NE) uptake and reduced basal and evoked neuronal NE release. Changes in NE uptake and release are generally associated to modifications in the synthesis and/or turnover of the amine. On this basis, the aim of the present work was to study ANF effects in the rat hypothalamus on the following processes: endogenous content, utilization and turn-over of NE; tyrosine hydroxylase (TH) activity; cAMP and cGMP accumulation and phosphatidylinositol hydrolysis. Results showed that centrally applied ANF (100 ng/microl/min) increased the endogenous content of NE (45%) and diminished NE utilization. Ten nM ANF reduced the turnover of NE (53%). In addition, ANF (10 nM) inhibited basal and evoked (with 25 mM KCl) TH activity (30 and 64%, respectively). Cyclic GMP levels were increased by 10 nM ANF (100%). However, neither cAMP accumulation nor phosphatidylinositol breakdown were affected in the presence of 10 nM ANF. The results further support the role of ANF in the regulation of NE metabolism in the rat hypothalamus. ANF is likely to act as a negative putative neuromodulator inhibiting noradrenergic neurotransmission by signaling through the activation of guanylate cyclase. Thus, ANF may be involved in the regulation of several central as well as peripheral physiological processes such as cardiovascular function, electrolyte and fluid homeostasis, endocrine and neuroendocrine synthesis and secretion, behavior, thirst, appetite and anxiety that are mediated by central noradrenergic activity.     

Modulation of depolarization stimulated 45Ca2+ uptake in cultured neuronal cells by calcium channel activators and antagonists

The effect of dihydropyridine calcium agonists and antagonists on 45Ca2+ uptake into primary neuronal cell cultures was investigated. K+ stimulated neuronal 45Ca2+ accumulation in a concentration dependent manner. This effect was further enhanced by the calcium agonists Bay K 8644 and (+)-(S)-202-791 with EC50 values of 21 nM and 67 nM respectively. The calcium antagonists PN 200-110 and (-)-(R)-202-791 inhibited Bay K 8644 (1 microM) stimulated uptake with IC50 values of 20 nM and 130 nM respectively. 45Ca2+ efflux from neuronal cells was measured in the presence and absence of Na+. Efflux occurred at a much greater rate from cells incubated in the presence of Na+, indicating the existence of an active Na+/Ca2+ exchanger in these neurons. The data suggests that voltage sensitive calcium channels of these neurons are sensitive to dihydropyridines and thus that dihydropyridine binding sites have a functional role in these neuronal cultures.     

Functional expression of the norepinephrine transporter in cultured rat astrocytes

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.     

The effect of desmethylimipramine on the metabolism of norepinephrine

Eleven normal volunteers were given an acute and two chronic doses of desipramine (DMI). The plasma norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), and dihydroxyphenylglycol (DHPG) concentrations were measured before and during drug administration. DMI reduced plasma concentrations of MHPG by 13% and DHPG by 17%. After two weeks of drug administration, the MHPG/NE ratio was reduced, and there was a significant negative correlation with the concurrent drug concentration. These results suggest that DMI: (1) reduces the turnover of NE; and (2) diminishes the oxidative deamination of NE. In addition, the drug concentration response relationship indicates that the effects of uptake inhibition may not be maximal until concentrations in the apparent therapeutic range are achieved.     

B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Vatta, M. S., M. F. Presas, L. G. Bianciotti, M. Rodriguez–fermepin, R. Ambros and B. E. Fernandez. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of ³H-NE. Experiments were carried out in rat adrenal medulla slices incubated “in vitro.” Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

dl-N-methyl-3-(o-methoxyphenoxy)-3-phenylpropylamine hydrochloride, Lilly 94939, a potent inhibitor for uptake of norepinephrine into rat brain synaptosomes and heart.

dl-N-Methyl-3-(o-methoxyphenoxy)-3-phenylpropylamine hydrochloride, Lilly 94949, is a potent inhibitor for uptake of norepinephrine (NE) into synaptosomes of rat brain with inhibitor constant (Ki) value of 1.8 × 10⁻⁷M. Lilly 94939 profoundly reduces the $\text{in vivo}$ accumulation of radioactivity from labeled NE in heart with ED₅₀ value of 1.5 mg/kg i.p. The inhibitory effects of the compound in synaptosomes and heart are most profound within 15 min of an intraperitoneal injection of Lilly 94939 at 10 mg/kg but much deminished at the 4th hr. These properties are in great contrast with its trifluoromethyl analog, Lilly 110140, which has previously been reported as a selective inhibitor of serotonin uptake in synaptosomes and without any effect on the accumulation of radioactivity from labeled NE in heart.     

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.     

Plasma 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers.

The usefulness of the plasma concentrations of two major metabolites of norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release was investigated. The potent alpha 2-adrenoceptor agonist, dexmedetomidine, induced only about 15% maximal reductions in the metabolite concentrations, in spite of almost total inhibition of neuronal NE release, as evidenced by 90% reductions in plasma NE concentrations. Similarly, administration of the alpha 2-adrenoceptor antagonist atipamezole was followed by only small increases in plasma DHPG and no change in MHPG levels, in spite of almost six-fold, albeit short-lasting, increases in plasma NE. In contrast, a single dose of the reversible monoamine oxidase type A (MAO-A) inhibitor moclobemide reduced plasma DHPG levels by 78% and MHPG levels by 51%. It is concluded that the plasma concentrations of DHPG and MHPG are largely determined by intraneuronal, MAO-A-dependent metabolism of NE, and do not accurately reflect acute alterations in neuronal NE release. The concentration of NE in venous plasma is clearly a more sensitive indicator of alpha 2-adrenoceptor-mediated regulation of NE release.     

3H]adrenaline release from hypothalamic synaptosomes and its modulation by clonidine: effects of chronic antidepressant drug regimens

[3H]Adrenaline ([3H]ADR, 40 nM) was accumulated by rat hypothalamic synaptosomes (P2) more rapidly and in significantly greater amounts than by similar preparations from cerebral cortex. There was no significant difference between these two tissues in the rate or amount of [3H]noradrenaline ([3H]NA, 40 nM) accumulation. Talusupram (10 microM), maximally inhibited the uptake of [3H]ADR into hypothalamic synaptosomes by 60%. Nomifensine further inhibited uptake by 14%. From these observations it was concluded that some [3H]ADR was accumulated into non adrenergic neuronal terminals. The effects of desipramine (DMI, 10 mg/kg/day and clorgyline (1 mg/kg/day) administration for 28 days on K+-evoked release of [3H]ADR was investigated using superfused hypothalamic synaptosomes. After both chronic antidepressant drug regimens, total [3H]ADR release (spontaneous + evoked) was significantly reduced. Evoked release of [3H]ADR (by KCl, 16 mM) was significantly reduced after the DMI but not the clorgyline regimens. Presynaptic alpha 2-adrenoceptor function in the hypothalamus was assessed during superfusion by measuring the reduction in K+-evoked release of [3H]ADR caused by clonidine (1 microM). The attenuating effects of clonidine on [3H]ADR release (42% in untreated controls and 36% after chronic clorgyline) was diminished (to 4%) after chronic DMI administration. Alpha 2 adrenoceptor numbers in the rat hypothalamus were not significantly changed after clorgyline or DMI administration, suggesting that the functional subsensitivity seen in synaptosomes after DMI, may not be related to alpha 2 adrenoceptor down regulation.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Stimulation of Na+,K+-ATPase activity in certain membranes of the rat central nervous system (CNS) by acute administration of desipramine (DMI)

1. The activities of ATPase in rat CNS were studied 3 hr after administration of the noradrenaline uptake inhibitor, desipramine (DMI: 10 mg.kg-1, i.p.). Na+K+-ATPase activity significantly increased after DMI in the whole particulate from hypothalamus and mesencephalus but no changes in frontal cortex or in pons-medulla oblongata areas were found. This increase was prevented when the animals were pretreated with the noradrenergic neurotoxic N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). 2. Purified membrane fractions from hypothalamus were obtained by differential and sucrose gradient centrifugation (0.8-1.2 M sucrose). It was observed that after DMI, Na+,K+-ATPase activity increased only in the membranous fraction lying at 0.9 M sucrose. 3. Mg2+- or Ca2+-ATPase activities were not modified by DMI treatment. 4. Citalopram, a specific serotonergic uptake inhibitor, did not affect ATPase activities. 5. The results obtained could indicate that DMI acute administration selectively stimulates Na+,K+-ATPase activity of certain membranes of the CNS after an increase in the concentration of the noradrenergic neurotransmitter in the synaptic gap.     

Selective expression of high-affinity uptake of catecholamines by transiently catecholaminergic cells of the rat embryo: studies in vivo and in vitro

Transient expression of catecholaminergic phenotypic traits is a widespread phenomenon during embryonic development in mammals, occurring in cells of the embryonic gut mesenchyme, in ventrolateral portions of the neural tube, cells of cranial sensory and dorsal root ganglia, and in the embryonic pancreas. In the current study the manifestation of the catecholamine (CA) phenotype in these populations has been further defined. Specifically, the existence of the high-affinity uptake process for CAs in these populations has been investigated. By combining the techniques of radioautography following accumulation of [3H]norepinephrine (3H-NE) and [3H]dopamine (3H-DA) with immunohistochemical detection of tyrosine hydroxylase (T-OH), it has been possible to demonstrate simultaneously CA accumulation by T-OH-positive gut cells. Uptake of 3H-NE was first detected in T-OH-positive cells of the gut on gestational day 12.5 (E12.5). By contrast, T-OH immunoreactivity was first detected on E11.5. By E13.5 virtually every T-OH-positive cell oral to the umbilical flexure was radioautographically labeled. Uptake at E13.5 displayed Michaelis-Menten saturation kinetics, had a Vmax of 35 fmole/gut/min, a Km of 1.45 microM, was blocked by desmethylimipramine (DMI), and by incubation at 4 degrees C. On subsequent gestational days, silver grains marking areas of amine concentration were found increasingly over T-OH-negative cells. A similar pattern of uptake was found in guts which had been grown in organotypic tissue culture for the purpose of eliminating extrinsic sympathetic innervation. T-OH-positive gut cells also accumulated 3H-DA. Concentration of 3H-DA was blocked by both benztropine and DMI suggesting that accumulation had properties common to both NE and DA systems. By contrast to cells of the gut, accumulation of CAs was not a property of transiently T-OH-positive cells in other locations. Therefore, specific, high-affinity uptake and retention of CAs is an additional property of transiently catecholaminergic gut cells. Appearance of CA synthetic enzymes precedes the appearance of the CA storage process in cells of the gut. Persistence of the uptake process after the loss of detectable T-OH suggests continued viability of the population. The absence of CA accumulation by other T-OH-positive cells suggests basic molecular differences among the various populations.     

CALCIUM METABOLISM IN ISOLATED BRAIN CELLS AND SUBCELLULAR FRACTIONS

Abstract— The accumulation of calcium ions by brain mitochondria and microsomes and by fractions containing neuronal or glial cells has been studied in vitro with techniques involving ⁴⁵Ca and ultramicro-flame photometry. ATP and substrate-supported calcium accumulation by brain mitochondria was of the same magnitude as for mitochondria from other organs. Brain microsomes accumulated calcium approximately 15 times less than brain mitochondria. Variations in Na⁺/K⁺ ratios and in ATP/ADP ratios had a more marked influence on microsomal uptake than on mitochondrial uptake. The passive Ca²⁺ binding by glial cells was higher than neuronal perikarya and synaptosomes. Also the calcium accumulation ability in cell suspensions was slightly higher for glial cells as compared to neuronal perikarya. The calcium uptake by glial cells was stimulated by high external K⁺ concentration, which also was the case for nerve endings. The uptake in neuronal perikarya was unaffected by variations in K⁺ concentration. A comparison between neuronal and glial mitochondria showed that both reach a steady state level of similar magnitude, but that the rate of initial accumulation was greater for glial mitochondria. A high glial calcium accumulation was also observed for the microsomal fraction.     

Uptake and accumulation in vitro of 3H-noradrenaline in adrenergic nerves of human atrium

Summary The adrenergic innervation and the in vitro uptake of ³H-noradrenaline has been investigated in human atrial tissue slices from patients undergoing thoracic surgery. The atrial appendage was richly innervated, but the density of the adrenergic nerve plexus varied considerably between different tissues examined. The nerve terminals were of characteristic varicose appearance, running singly or in bundles along the long axis of the muscle fibers. The nerve fibers seemed to penetrate in between the muscle cells. The distribution and appearance of the adrenergic nerves were quite similar to those described in earlier investigations of heart tissue from other species. The uptake and accumulation of ³H-noradrenaline in vitro increased with increasing concentration in the medium and with time, and the uptake process could efficiently be blocked by desmethylimipramine (DMI), a potent inhibitor of the uptake mechanism located at the axonal membrane, the so called membrane pump. There was a true accumulation of ³H-noradrenaline in the atrial tissue during the incubation, compared to the medium. The metabolism of ³H-noradrenaline during the incubation has also been studied. The data presented speak in favour of the view that the adrenergic nerves of human atria possess an efficient uptake accumulation mechanism for noradrenaline.Abbreviations used DMI desmethylimipramine - NA noradrenaline - NM normetanephrine     

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.