The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.     

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.     

Regulation of interleukin 2 synthesis by cAMP in human T cells

T cell activation requires two initial signals that first lead to the expression of interleukin 2 (IL 2) receptors and the initiation of IL 2 synthesis and then to T cell proliferation. Jurkat T lymphoma cells have been shown to be a good model for studying IL 2 synthesis because these cells also require two signals for activation. The first signal can be provided by the lectin phytohaemagglutinin (PHA), and the second one by the phorbol ester, 12-o-tetradecanoylphorbol 13-acetate (TPA). The regulation of IL 2 synthesis in Jurkat cells, however, is unclear, and the present study deals with the role of cAMP on IL 2 synthesis. In Jurkat cells, IL 2 synthesis appears to be highly regulated by the activity of adenylate cyclase. This was demonstrated by using different means to increase intracellular cAMP level, namely by using permeant cAMP analogs, using the activator of adenylate cyclase, forskolin, using the activator of the alpha subunit of the stimulatory GTP binding protein cholera toxin, and using inhibitors of phosphodiesterase. In addition, prostaglandins E1 and E2 were shown to bind specifically to Jurkat cells, to induce a rise in intracellular cAMP level, and to markedly decrease IL 2 synthesis. All together, these results suggest that in T lymphocytes, the prostaglandin E2 receptor is linked to adenylate cyclase through a GTP binding protein and regulates the production of IL 2 by controlling the intracellular cAMP level.     

Cloning and characterization of a Drosophila tyramine receptor.

Receptors for biogenic amines such as dopamine, serotonin and epinephrine belong to the family of receptors that interact with G proteins and share a putative seven transmembrane domain structure. Using a strategy based on nucleotide sequence homology between the corresponding genes, we have isolated Drosophila cDNA clones encoding a new member of the G protein-coupled receptor family. This protein exhibits highest homology to the human alpha 2 adrenergic receptors, the human 5HT1A receptor and a recently cloned Drosophila serotonin receptor. The corresponding mRNA is found predominantly in adult Drosophila heads. Membranes from mammalian cells expressing this receptor displayed high affinity binding sites for [3H]yohimbine, an alpha 2 adrenergic receptor antagonist (Kd = 4.45 x 10(-9) M). Tyramine was the most efficient of the putative Drosophila neurotransmitters at displacing [3H]yohimbine binding (EC50 = 1.25 x 10(-6) M). Furthermore tyramine induced an inhibition of adenylate cyclase activity in NIH 3T3 cells expressing this receptor. The Drosophila tyramine receptor that we have isolated might therefore be an invertebrate equivalent of the mammalian alpha 2 adrenergic receptors.     

Intracellular angiotensin II inhibits heterologous receptor stimulated Ca2+ entry.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.     

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.     

Agonist-stimulated phosphatidylinositol 4,5-bisphosphate metabolism in the nervous system

Phosphatidylinositol 4,5-bisphosphate has recently gained prominence as the central component of a receptor transduction process which generates inositol 1,4,5-trisphosphate and diacylglycerol in stimulated cells. Both of these products of phospholipid metabolism have intracellular second messenger functions with diacylglycerol formation leading to activation of protein kinase C and inositol 1,4,5-trisphosphate stimulating Ca²⁺ release from intracellular stores in the endoplasmic reticulum. There is mounting evidence that the phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate is coupled to activated receptors by a guanylnucleotide binding protein, analogous to Ns and Ni which couple stimulatory and inhibitory hormone receptors to adenylate cyclase. Most of the key elements of this signalling mechanism have been found in the nervous system and so too has an entirely novel and unexpected inositol phosphate ester, inositol 1,3,4,5-tetrakisphosphate, whose function is not yet known. Phosphatidylinositol 4,5-bisphosphate breakdown, detected as the accumulation of inositol phosphates in agonist-stimulated nervous tissue preparations, is a functional response that has been useful in assessing the relevance of receptors identified by radioligand binding assays, and which provides an essential link between receptor occupation and responses such as neurotransmitter release and modulation of neuronal excitability.     

Multiple mechanisms of serotonergic signal transduction

In this article we review serotonergic signal transduction mechanisms in the central and peripheral nervous systems and in a variety of target organs. The various classes of pharmacologically defined serotonergic receptors are coupled to three major effector systems: (1) adenylate cyclase; (2) phospholipase C mediated phosphoinositide (PI) hydrolysis and (3) ion channels (K+ and Ca++). Long term occupancy of serotonergic receptors also appears to induce alterations in mRNA and protein synthesis. For all three types of signal transduction there is evidence accumulating which suggests the involvement of guanine nucleotide regulatory proteins. Recent findings suggest that the distinct types of pharmacologically defined serotonergic receptors (5HT1A, 5HT1B, 5HT1c, 5HT2) may be coupled to one or more signal transduction systems. Thus, 5HT1 receptors may both activate and inhibit adenylate cyclase and increase K+-ion conductance in the hippocampus. 5HT2 receptors which activate PI hydrolysis in the brain, both open voltage-gated calcium channels and activate PI metabolism in certain smooth muscle preparations. Thus, each class of serotonergic receptor may be linked to one or more distinct biochemical transduction mechanisms. The possibility is raised that selective agonists and antagonists might be developed which have specific effects on a particular receptor-linked effector system.     

Hormonal regulation of phosphatidylinositol breakdown

Cyclic AMP and Ca2+ are intracellular mediators of hormone action. Catecholamines interact with beta adrenoceptors to activate adenylate cyclase or with alpha 2 adrenoceptors to inhibit adenylate cyclase. Alpha 1 adrenoceptor activation results in elevation of cytosol Ca2+ and an increased breakdown of phosphatidylinositol. In blowfly salivary glands, 5-hydroxytryptamine (5-HT) interacts with beta type receptors resulting in adenylate cyclase activation while alpha type receptors are involved in phosphatidylinositol breakdown and elevation of cytosol Ca2+. The link between Ca2+ mobilization and phosphatidylinositol breakdown remains to be established but breakdown of the receptor-regulated pool of phosphatidylinositol is not secondary to the rise in Ca2+. Direct addition of 5-HT to cell-free homogenates of blowfly salivary glands results in activation of phosphatidylinositol breakdown in the absence of Ca2+. In rat liver plasma membrane preparations, vasopressin increases phosphatidylinositol breakdown in the absence of Ca2+ or cytosol if deoxycholate is present. The data do not indicate whether hormone activation increases the availability of substrate to enzymatic hydrolysis or activates phospholipase C. However, they demonstrate that hormones directly accelerate phosphatidylinositol breakdown.     

Transformation of BALB/3T3 cells with EJ/T24/H-ras oncogene inhibits adenylate cyclase response to beta-adrenergic agonist while increases muscarinic receptor dependent hydrolysis of inositol lipids

Balb/3T3 murine fibroblasts transformed by transfection with the EJ/T24 human bladder carcinoma oncogene were assayed in terms of adenylate cyclase response and hydrolysis of polyphosphoinositides dependent on specific agents. Transformed cells were much less responsive to beta-adrenergic agonists in rising cAMP than normal cells. They are instead much more sensitive to muscarinic receptor agonists, inducing a rapid intracellular accumulation of inositol phosphates. These results suggest that the functional alteration of the cell membrane caused by the product of the point mutated H-ras oncogene concerns in 3T3 fibroblasts both inhibitory and stimulatory effects, respectively on adenylate cyclase and phosphoinositide-phosphodiesterase.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor

5'-(N-Ethyl)carboxamidoadenosine (NECA), an analog of adenosine, transiently stimulated a rat tumor mast cell (RBL-2H3 cells) to cause a release of inositol phosphates and an increase in levels of Ca2+ in the cytosol. It failed, however, to stimulate a sustained uptake of 45Ca2+ or secretion. The effects of other agents that act on P1- or P2-purinergic receptors suggested that NECA and other adenosine agonists acted via a novel subtype of adenosine membrane receptor. Although the order of potency of agonists was characteristic of A2-adenosine receptors, there was no indication of the involvement of adenylate cyclase, and antagonists such as isobutylmethylxanthine, 8-phenyltheophylline, and 8-p-sulfophenyltheophylline inhibited the responses to either NECA or antigen. The fact that stimulation of inositol phospholipid hydrolysis by NECA in washed, permeabilized RBL-2H3 cells was blocked by pertussis toxin as well as by cholera toxin suggested instead that the NECA-sensitive receptor activated phospholipase C via a G-protein. In contrast to NECA, antigen stimulation resulted in a pertussis toxin-resistant, sustained hydrolysis of inositol phospholipids, increases in free intracellular Ca2+, accelerated influx of 45Ca2+, and secretion from RBL-2H3 cells. In combination with NECA, all responses to antigen were markedly enhanced, and the enhancement was selectively blocked by pertussis toxin. The ability of antigen, but not NECA, to provoke secretion may be dependent primarily on the sustained activation of a cholera toxin-sensitive Ca2+ influx pathway that serves to amplify stimulatory signals for secretion. These studies also suggested that phospholipase C could be activated through different G-proteins via different receptors within the same cell.     

Multiple signal transduction pathways mediated by 5-HT receptors

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.     

Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis.

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Using biosensors to detect the release of serotonin from taste buds during taste stimulation

CHO cells transfected with high-affinity 5HT receptors were used to detect and identify the release of serotonin from taste buds. Taste cells release 5HT when depolarized or when stimulated with bitter, sweet, or sour tastants. Sour- and depolarization-evoked release of 5HT from taste buds is triggered by Ca2+ influx from the extracellular fluid. In contrast, bitter- and sweet-evoked release of 5HT is triggered by Ca2+ derived from intracellular stores.     

Phorbol 12-myristate 13-acetate (TPA) blocks CD3-mediated Ca2+ mobilization in Jurkat T cells independently of protein kinase C activation

Stimulation of Jurkat T cells with antibodies against the T cell receptor/CD3 complex induces a rise in the intracellular concentration of Ca2+ within seconds. The inositol phosphate-dependent Ca2+ mobilization induced by OKT3 was completely abrogated when Jurkat cells were pretreated for 1 min with the phorbol 12-myristate 13-acetate TPA (10nM), a concentration which activates protein kinase C (PKC). The effects of TPA on the Ca2+ fluxes were insensitive to treatment of the cells with known PKC inhibitors (H-7 and staurosporin) under conditions where the PKC-mediated phosphorylation was blocked. Furthermore, another activator of PKC, mezerein, inhibited the Ca2+ signal induced by OKT3. This inhibition, however, could completely be reversed by pretreatment with H-7 or staurosporine. We conclude that the TPA-mediated inhibition of Ca2+ fluxes in Jurkat T cells largely acts through a PKC-independent pathway.