Increased Phosphatidylinositol Turnover Research Articles

Muscarinic receptors of the albino rabbit ciliary process

Muscarinic receptor binding sites were identified in membranes prepared from albino rabbit ciliary processes, using the muscarinic antagonist [ 3H] l-quinuclidinyl benzylate as the radioligand. Analysis of saturation binding experiments demonstrated that [ 3H] l-quinuclidinyl benzylate bound to an apparent homogeneous population of binding sites with a K d value of 6:4 p m and a B max value of 155 fmol mg −1 protein. Seventy percent (70%) of binding sites showed high affinity for pirenzepine, i.e. belonged to the M 1 subtype. In contrast, AF-DX 116 was unable to discriminate between subtypes of muscarinic binding sites in this tissue. Carbachol caused a dose-dependent increase in phosphatidylinositol turnover (EC 50 = 154 μ m) in ciliary processes. A maximum stimulation of 652% of basal activity was obtained following a 45 min incubation with 10 m m carbachol. The potency of muscarinic antagonists to block the carbachol-induced response was comparable to that found for M 1 receptors in other tissues. Oxotremorine and pilocarpine behaved like partial agonists in this assay. The carbachol-induced increase in phosphatidylinositol turnover was also observed in a suspension of epithelial cells from ciliary processes and it was blocked by atropine; thus, indicating the presence of muscarinic receptors functionally coupled to phosphatidylinositol turnover in these cells.

Mitogenesis by serum and PDGF is independent of PI degradation and PKC in VSMC

Capacities of serum, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) on phosphatidylinositol (PI) degradation and cell growth were compared in cultured vascular smooth muscle cells (VSMC) from rat aorta. The role of protein kinase C (PKC) in growth control was also evaluated using polymixin B, a selective inhibitor of PKC. Both dialyzed and nondialyzed fetal bovine serum (FBS) in concentrations from 2 to 20% stimulated [3H]thymidine incorporation into DNA and cell growth without producing corresponding increases in PI turnover. Moreover, both PDGF (40-160 ng/ml) and FGF (6.25-150 ng/ml) also stimulated mitogenesis, but PDGF was more effective although less potent. Mitogenic amounts of PDGF did not stimulate PI turnover, whereas a maximally mitogenic amount of FGF (50 ng/ml) did produce a slight increase. Polymixin B inhibited PKC activity (IC50, 32 microM) from these cells but failed to suppress DNA synthesis produced by 10% FBS or PDGF (50 ng/ml). However, it did suppress that by FGF (50 ng/ml). Angiotensin II (10(-11)-10(-7) M) and phorbol 12,13-dibutyrate (PDB, 1-20 nM) were not mitogenic in the presence or absence of insulin (10 micrograms/ml) or the calcium ionophore A23187 (0.25-4 microM), under serum-free conditions. Instead, PDB inhibited mitogenesis of cells maintained under 0.2% FBS or stimulated with insulin (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium mobilization and phosphatidylinositol turnover in vas deferens smooth muscle of diabetic rats

The study concerned Ca 2+ channels that are receptor-operated by norepinephrine (NE) and mediate hyper-reactivity of vas deferens smooth muscle from rats with streptozotocin (STZ)-induced diabetes, and the mediatory responses of these channels, such as tension development, Ca 2+ uptake and phosphatidylinositol (PI) turnover. The contractile responses induced by adrenoceptor agonists were significantly greater in diabetic rat vas deferens than in the controls. A greater Ca 2+ uptake was induced by 10 −5 M NE in strips from diabetic rats than in the controls. The uptake of Ca 2+ was completely inhibited by 10 −6 M prazosin but not by 10 −5 M verapamil. Enhancement of Ca 2+ release by 10 −5 M NE was faster and greater in diabetic muscles than in the controls. The accumulation of [ 3H]inositol phosphates was increased 4-fold in the controls and 7-fold in diabetic muscles by 10 −5 M NE. This increase was completely inhibited by 10 −6 M prazosin but not by 10 −6 M yohimbine. The data suggest that vas deferens smooth muscle hyper-reactivity in diabetic rats is due to increased PI turnover mediated by α 1-adrenoceptors, to the release of intracellular bound Ca 2+ and to an increase of Ca 2+ uptake through receptor-operated Ca 2+ channels.

Distinct pathways mediate transcriptional regulation of platelet-derived growth factor B/c-sis expression.

The biochemical mechanisms responsible for regulating cellular platelet-derived growth factor expression are incompletely understood. Our previous studies have shown that platelet-derived growth factor B/c-sis mRNA levels are induced in human renal microvascular endothelial cells by either thrombin or transforming growth factor (TGF-beta), while exposure to agents which elevate cAMP levels blocks the induction responses. The current studies use combined transcription run-off and message decay rate experiments to show greater than 3-fold increases in rate of transcription after stimulation with either thrombin or TGF-beta. c-sis message has a 70-90-min half-life under basal conditions that is effectively unaltered by thrombin or TGF-beta. Forskolin does not decrease the stability of c-sis mRNA, although it attenuates transcription increases seen with inducing agents. TGF-beta induction of c-sis transcription is mediated independent of the protein kinase C (Ca2+- and phospholipid-dependent enzyme)-mediated responses to phorbol ester, as it remains intact following down-regulation of protein kinase C response; TGF-beta and phorbol elicit additive induction. Inhibitory effects of cAMP upon transcription act distal to early thrombin-receptor-coupled increases in phosphatidylinositol turnover and are capable of turning off TGF-beta-activated transcription after activation has been established. Both inducing and suppressing agents alter endothelial platelet-derived growth factor B/c-sis mRNA expression dominantly through effects upon rates of transcription, cAMP suppression of transcription is dominant, and TGF-beta and phorbol esters mediate induction of transcription through distinct pathways.

The release of bombesin-like peptides from small cell lung cancer cells.

High levels of BN/GRP are present in classic SCLC and lung carcinoids, whereas BN immunoreactivity is absent in variant SCLC, adenocarcinoma, large cell carcinoma, squamous cell carcinoma, and mesothelioma cell lines. BN-like peptides are secreted from classic SCLC into the tissue culture medium. The secretion rate of BN-like peptides from cell line NCI-H345 was increased 3-fold by VIP (1 microM). Also, VIP increased the cAMP levels in cell line NCI-H345 by an order of magnitude. Therefore, SCLC cells have functional VIP receptors which regulate the secretion of BN-like peptides. Also, SRIF (100 nM) inhibits the VIP-stimulated increase in cAMP levels and secretion rate of BN-like peptides from SCLC cells. Because BN stimulates colony formation, VIP and/or SRIF may be able to alter the growth of SCLC cells. BN-like peptides are secreted from SCLC cells into the plasma. The levels of BN immunoreactivity in the plasma of SCLC patients with extensive disease is 2- to 40-fold greater than that of patients with limited disease. Secretin infusion into patients with extensive disease produces a transient increase (7-fold) in the plasma concentration of BN-like peptides. BN-like peptides are also present in the CSF of SCLC patients. When released from SCLC cells, BN-like peptides may interact with cell surface receptors. [Tyr4]BN binds with high affinity (Kd = 0.5 nM) to a single class of sites (1500/cell) on cell line NCI-H345. The carboxyl terminus of BN or GRP is essential for high-affinity binding activity. BN-like peptides elevate cytosolic Ca2+ levels as a result of increased phosphatidylinositol turnover. The putative BN receptor antagonist [D-Arg1, D-Pro2, D-Trp7,9, Leu11]substance P inhibits high-affinity [Tyr4]BN binding, the ability of BN to elevate cytosolic Ca2+ levels, and colony formation of SCLC cells. Therefore, BN receptor antagonists may serve as useful agents to inhibit the growth of SCLC.

A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line

The human small cell lung carcinoma (SCLC) cell line NCI-H345 constitutively produces gastrin-releasing peptide (GRP), a peptide homologous to the mitogen bombesin. In addition, NCI-H345 cells express bombesin receptors and respond to bombesin with rapid activation of phospholipase C and mobilization of intracellular Ca2+. Treatment of NCI-H345 cells with a novel potent bombesin receptor antagonist [Leu13-psi-CH2NH-Leu14]bombesin blocked the increase in phosphatidylinositol turnover and cytoplasmic free Ca2+ ([Ca2+]i) stimulated by bombesin. Furthermore [Leu13-psi-CH2NH-Leu14]bombesin inhibited NCI-H345 colony formation in defined semisolid medium in the absence of exogenous GRP. The rapid, hormone-induced accumulation of inositol(1,4,5)trisphosphate was markedly more sensitive to antagonist inhibition than the hormone-induced Ca2+ transient, the sustained accumulation of inositol monophosphates, or colony formation in soft agarose. These data demonstrated inhibition of transmembrane signals associated with autocrine growth control in SCLC by a novel peptide receptor antagonist.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells.

The effect of prostaglandins (PG) on free cytosolic calcium concentrations [( Ca2+]i) and cAMP levels was studied in the osteosarcoma cell line UMR-106. PGF2 alpha and PGE2, but not 6-keto-PGF1 alpha, induced an increase in [Ca2+]i which was mainly due to Ca2+ release from intracellular stores. The EC50 for PGF2 alpha was approximately 7 nM, whereas that for PGE2 was approximately 1.8 microM. Maximal doses of PGF2 alpha increased [Ca2+]i to higher levels than PGE2. Both active PGs also stimulated phosphatidylinositol turnover in UMR-106 cells. The effects of the two PGs were independent of each other and appear to involve separate receptors for each PG. PGE2 was a very potent stimulator of cAMP production and increased cAMP by approximately 80-fold with an EC50 of 0.073 microM. PGF2 alpha was a very poor stimulator of cAMP production; 25 microM PGF2 alpha increased cAMP by 5-fold. The increase in cellular cAMP levels activated a plasma membrane Ca2+ channel which resulted in a secondary, slow increase in [Ca2+]i. High concentrations of both PGs (10-50 microM) inhibited this channel independent of their effect on cAMP levels. Pretreatment of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate inhibited the PG-mediated increase in phosphatidylinositol turnover and the increase in [Ca2+]i. However, pretreatment with 12-O-tetradecanoyl-13-acetate had no effect on the PGE2-mediated increase in cAMP. The latter finding, together with the dose responses for PGE2-mediated increases in [Ca2+]i and cAMP levels, suggests the presence of two subclasses of PGE2 receptors: one coupled to adenylate cyclase and the other to phospholipase C. With respect to osteoblast function, the cAMP signaling system is antiproliferative, whereas the Ca2+ messenger system, although having no proliferative effect by itself, tempers cAMP's antiproliferative effect.

Comparison of the effects of some muscarinic agonists on smooth muscle function and phosphatidylinositol turnover in the guinea‐pig taenia caeci

1. The effects of the muscarinic agonists acetylcholine (ACh), carbachol (CCh), AHR-602, and McN-A-343 on contractility and on inositol phosphate accumulation in the presence of lithium were compared in the taenia of the guinea-pig caecum. 2. Compared to CCh, ACh was a full agonist for contraction but AHR-602 and McN-A-343 were partial agonists producing 80-85% of the maximal response to CCh. Similar to previous findings with CCh, tonic contractions produced by AHR-602 and McN-A-343 were less sensitive to inhibition by nifedipine or verapamil than tonic contractions to ACh. 3. CCh and ACh produced similar increases in inositol phosphate accumulation and the effect of CCh (0.1 mM) was inhibited by atropine (IC50 8.5 nM) and pirenzepine (IC50 450 nM). The accumulation of inositol phosphates in the presence of AHR-602 or McN-A-343 was not significantly different (P greater than 0.05) from basal levels. 4. A concentration of 0.2 mM AHR-602 produced a parallel shift of the concentration-response curve to CCh on inositol phosphate accumulation. The IC50 value for inhibition of CCh (0.1 mM) was greater than 50 fold higher than the EC50 value for contraction produced by the partial agonist. McN-A-343 (20 microM) produced a flattening of the concentration-response curve to CCh for inositol phosphate accumulation. 5. The results suggest that the increase in phosphatidylinositol turnover produced by muscarinic agonists, like the contractile response, involves an M2-muscarinic receptor. AHR-602 and McN-A-343 are partial agonists for the contractile response and while producing no significant increase in phosphatidylinositol turnover inhibit the response to CCh.

Prostanoid responses of bovine adrenal medullary cells: lack of effect of opioids

The effects of opioid compounds on catecholamine (CA) secretion and phosphatidylinositol turnover induced by prostaglandins E 1 (PGE 1) and E 2 (PGE 2) in cultured bovine adrenal medullary cells have been studied. PGE 1 induced CA secretion at 100 nM and above. PGE 2 was more potent, inducing CA secretion at 1–10 nM. Both prostaglandins required extracellular calcium to induce CA release. Neither etorphine nor diprenorphine (1 nM-10 μM) affected CA secretion induced by 1 μM PGE 1 or 0.1 μM PGE 2. PGEP 1 produced a small increase in phosphatidylinositol turnover at 10 μM, but had no effect at lower concentrations. PGE 2 was effective at 1 and 10 μM. Etorphine and diprenorphine had no effect on phosphatidylinositol turnover induced by PGE 1 or PGE 2. The results indicate prostaglandins can facilitate CA secretion independently of their effects on phosphatidylinositol metabolism. They also indicate that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary cells to modify their responses to prostaglandins.

The phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg 2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.

Inhibition of phosphatidylinositol kinase in vascular smooth muscle membranes by adenosine and related compounds

Adenosine and 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of phosphatidylinositol in membranes prepared from aortic smooth muscle. The nucleosides did not affect the breakdown of phosphatidylinositol-4-phosphate. Under certain conditions, the membrane-bound phosphatidylinositol kinase phosphorylated exogenous phosphatidylinositol. The nucleosides inhibited the enzyme competitively with respect to magnesium-ATP and non-competitively with respect to phosphatidylinositol. Adenosine analogs modified in the ribose moiety were inhibitors with potencies comparable to that of adenosine, whereas adenine nucleotides and purine-modified adenosine analogs were much weaker inhibitors. Density gradient fractionation studies showed that phosphatidylinositol kinase is primarily associated with the sarcoplasmic reticulum. Vascular smooth muscle contraction is associated with increased phosphatidylinositol turnover. Inhibition of phosphatidylinositol kinase by intracellular adenosine may, therefore, be a factor involved in regulating vasodilation.

Muscarinic receptor subtypes in human and rabbit bladder

AbstractSubtypes of muscarinic receptors have been proposed by several investigators, based on differences in the potency of muscarinic agonists in various tissues. Development of selective muscarinic agonists and antagonists for the urinary bladder would be extremely useful since side effects of the presently used drugs often limit their clinical usefulness. Direct investigation of muscarinic receptor subtypes by radioligand binding techniques has recently become possible. The muscarinic antagonist pirenzepine distinguishes between a high affinity and low affinity binding site in several tissues. Results of the present investigation indicate that only the low affinity pirenzepine binding site is present in both human and rabbit bladder. Muscarinic receptor linked biochemical responses (increased phosphatidyl inositol turnover and inhibition of adenyl cyclase) were also consistent with the presence of only the low affinity pirenzepine binding receptor subtype in both human and rabbit bladder.

Modulation of phosphatidylinositol turnover by liposomes containing phosphatidylinositol

This chapter discusses the methods for preparing liposomes for coating (opsonizing) the liposomes with immunoglobulin M (IgM) antibodies and complement or opsonizing with concanavalin A (Con A) to promote phagocytosis and resulting increased phosphatidylinositol (PI) turnover, for obtaining and culturing mouse peritoneal macrophages, and for measuring the uptake of radiolabeled inositol into PI by macrophages in culture. The method involves stimulation of PI turnover by incubating macrophages with liposomes coated (opsonized) with complement. The C3b bound to the liposomes is recognized by the C3b receptor and this results in a greatly enhanced phagocytosis of liposomes. Complement-dependent phagocytosis of liposomes is associated with increased PI turnover. By using a monoclonal IgM antibody that recognizes liposomal phosphatidylinositol 4-phosphate (PIP), one is successful in suppressing increased PI turnover without suppressing complement-dependent phagocytosis. The chapter describes opsonization by complement. Opsonization by complement is achieved by first attaching IgM antibodies to a liposomal antigen on the surface of the liposomes and then exposing the liposomes to complement.

Stimulation of phosphatidylinositol turnover by concanavalin a is not sufficient to activate mouse thymocytes

Mouse thymocytes treated with the lectin Concanavalin A do not proliferate nor do they develop responsiveness to interleukin 2. Co-treatment with Concanavalin A and either lectin-activated splenocyte conditioned medium or phorbol ester caused increased interleukin 2 receptor expression and proliferation. Under these conditions, lectin alone stimulated a 3.4 fold increase in phosphatidylinositol turnover which was unaffected by the presence of conditioned medium. Phosphorylation of a 55 kD protein was stimulated in response to conditioned medium or phorbol ester, but not lectin. These results indicate that stimulation of phosphoinositide turnover is not sufficient to activate thymocytes, and suggest that costimulating factors activate a kinase which is distinct from protein kinase C, or alternatively, activate protein kinase C through a process which is not coupled to phosphoinositide turnover.

Changes in free cytosolic calcium and accumulation of inositol phosphates in isolated hepatocytes by [Leu]enkephalin.

Isolated hepatocytes from fed rats were used to study the effects of the opioid peptide [Leu]enkephalin on intracellular free cytosolic Ca2+ ([Ca2+]i) and inositol phosphate production. By measuring the fluorescence of the intracellular Ca2+-selective indicator quin-2, [Leu]enkephalin was found to increase [Ca2+]i rapidly from a resting value of 0.219 microM to 0.55 microM. The magnitude of this response was comparable with that produced by maximally stimulating concentrations of either vasopressin (100 nM) or phenylephrine (10 microM). The opioid-peptide-mediated increase in [Ca2+]i showed a dose-dependency comparable with the activation of phosphorylase, but it preceded the increase in phosphorylase alpha activity. Addition of [Leu]enkephalin to hepatocytes prelabelled with myo-[2-3H(n)]inositol resulted in a significant stimulation of inositol phosphate production. At 10 min after hormone addition, there were increases in the concentrations of inositol mono-, bis- and tris-phosphate fractions of 12-, 9- and 14-fold respectively. No effect was apparent on the glycerophosphoinositol fraction. The effect of 10 microM-[Leu]enkephalin on inositol phosphate production was significantly greater than that obtained with 10 microM-phenylephrine, but marginally smaller than that induced by 100 nM-vasopressin. However, at these concentrations all three agonists gave a comparable increase in [Ca2+]i and activation of phosphorylase a. These data provide evidence for [Leu]enkephalin acting via a mechanism involving a mobilization of Ca2+ as a result of increased phosphatidylinositol turnover.

Phosphatidylinositol liposomes opsonized by concanavalin a stimulate phosphatidylinositol turnover in macrophages

We have previously found that concanavalin A binds specifically to inositol and phosphatidylinositol. In the present study we demonstrate that binding of concanavalin A to liposomes containing phosphatidylinositol influences the uptake of such liposomes by macrophages. Although resident mouse peritoneal macrophages normally ingest liposomes only to a slight extent, attachment of concanavalin A to the liposomes caused a marked enhancement of phagocytosis. Furthermore induction of phagocytosis of concanavalin A-opsonized liposomes was associated with increased phosphatidylinositol turnover. We conclude that (a) concanavalin A can serve as an opsonizing agent for liposomes; (b) opsonization results from binding of concanavalin A to phosphatidylinositol on the liposomal surface; and (c) concanavalin A-induced phagocytosis is associated with increased phosphatidylinositol turnover in the macrophages.

A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover.

A novel serotonergic binding site, the 5-HT1C site, has been characterized recently in choroid plexus and several brain regions. The biochemical and physiological roles of this site have not been previously described. In this report we show that serotonin (5-hydroxytryptamine, 5-HT) stimulates phosphatidylinositol turnover in rat choroid plexus. The pharmacology of serotonin-stimulated phosphatidylinositol hydrolysis in choroid plexus was compared to the pharmacology in cerebral cortex, where this response is mediated by the serotonin 5-HT2 receptor. Serotonin increased phosphatidylinositol turnover in choroid plexus by 6-fold and in cerebral cortex by 2.5-fold. Serotonin was greater than 10-fold more potent in choroid plexus (EC50 = 46 nM) than in cerebral cortex (EC50 = 540 nM). The serotonin antagonists ketanserin, mianserin, and spiperone inhibited the response in the two tissues with different potencies. In cerebral cortex all three exhibited nanomolar affinities consistent with their potencies at the 5-HT2 site. In choroid plexus, however, the rank order (mianserin greater than ketanserin much greater than spiperone) and absolute potencies were consistent with binding to the 5-HT1C site. These data suggest that the 5-HT1C site in choroid plexus is a functional receptor that utilizes phosphatidylinositol turnover as its biochemical effector system.

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex.

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.