Somatostatin analog SMS 201995 inhibits proliferation in human leukemia T-cell line: relevance of the adenylyl cyclase stimulation

Octreotide SMS 201995 is a stable somatostatin (SRIF) analog with potent antiproliferative actions in numerous cell types including normal T lymphocytes. It is currently used in the clinical treatment of different malignancies. However, the possible beneficial actions of octreotide in T-cell leukemia have not been addressed before, although these cells express SRIF receptors. For instance, human leukemia Jurkat T cells have been shown to express a single SRIF receptor isotype: sst3 that can be pharmacologically targeted by octreotide. In this study, we therefore studied SMS 201995 effects on in vitro [(3)H-CH3]thymidine incorporation in Jurkat T cells. Our data show that octreotide inhibits the proliferation of Jurkat cells both in the absence and in the presence of mitogens. By contrast, SRIF28, an endogenous SRIF analog sharing with SMS 201995 an almost identical affinity for somatostatin sst3 receptors, increases [(3)H-CH3]thymidine uptake in both mitogen-activated and nonactivated cells. To assess the mechanisms of the opposite actions of these two analogs on leukemia T-cell proliferation, we next studied their effects on adenylyl cyclase activity in whole Jurkat cells. At least in the presence of mitogens, SMS 201995 significantly enhances the adenylyl cyclase activity whereas SRIF28 inhibits it. Taken together these data are in accordance with the current hypothesis according to which increase and decrease in cAMP production are required to allow the inhibition and stimulation of T-cell proliferation, respectively. They also point to a potential therapeutic benefit of SMS 201995 in the management of human T-cell leukemia.     

Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM]]; n = 4) of two somatostatin binding sites identified in control Jurkat cells (Ki1 = 78 +/- 3 pM and Ki2 = 12 +/- 4 nM [mean +/- SEM]; n = 4) was also observed. Almost identical results were obtained for phytohemagglutinin-activated human PBMC. In the absence of MV infection, two somatostatin binding sites were present (Ki1 = 111 +/- 31 pM and Ki2 = 17 +/- 2 nM [mean +/- SEM]; n = 2), whereas in MV-infected cells, only the high-affinity (Ki1 = 48 +/- 15 pM [mean +/- SEM]; n = 2) binding site remained. In addition, MV infection reinforced the inhibitory effects of SRIF on adenylyl cyclase activity, since maximal inhibition at 1 microM peptide was 11% +/- 4% in control cells versus 25% +/- 3% (P < 0.05) in infected Jurkat cells. Moreover, MV infection severely impaired the capacity of adenylyl cyclase to be activated directly (by forskolin) or indirectly (via Gs protein-coupled vasoactive intestinal peptide receptor). An assessment of [methyl-3H]thymidine incorporation showed that SRIF increased proliferative responses to mitogens only in control cells, not in MV-infected cells. Altogether, our data emphasize that MV-associated alteration of SRIF transduction appears to be related to the loss of SRIF-dependent increase of mitogen-induced proliferation.     

Somatostatin increases mitogen-induced IL-2 secretion and proliferation of human jurkat T cells via sst3 receptor isotype

The neuropeptide somatostatin (SRIF) modulates normal and leukemia T cell proliferation. However, neither molecular isotypes of receptors nor mechanisms involved in these somatostatin actions have been elucidated as yet. Here we show by using RT-PCR approach that mitogen-activated leukemia T cells (Jurkat) express mRNA for a single somatostatin receptor, sst3. This mRNA is apparently translated into protein since specific somatostatin binding sites (K_I1 = 78 ± 3 pM) were detected in semipurified plasma membrane preparations by using ¹²⁵I-Tyr¹-SRIF14 as a radioligand. Moreover, somatostatin inhibits adenylyl cyclase activity with similar efficiency (IC₅₀ = 23 ± 4 pM) thus strongly suggesting a functional coupling of sst3 receptor to this transduction pathway. The involvement of sst3 receptor in immuno-modulatory actions of somatostatin was assessed by analysis of neuropeptide effects on IL-2 secretion and on proliferation of mitogen-activated Jurkat cells. Our data show that in the concentrations comprised between 10 pM and 10 nM, somatostatin potentiates IL-2 secretion. This effect is correlated with somatostatin-dependent increase of Jurkat cell proliferation since the EC₅₀ concentrations for both actions were almost identical (EC₅₀ = 22 ± 9 pM and EC₅₀ = 12 ± 1 pM for IL-2 secretion and proliferation, respectively). Altogether, these data strongly suggest that in mitogen-activated Jurkat cells, somatostatin increases cell proliferation through the increase of IL-2 secretion via a functional sst3 receptor negatively coupled to the adenylyl cyclase pathway. J. Cell. Biochem. 68:62–73, 1998. © 1998 Wiley-Liss, Inc.     

Somatostatin inhibits follicle-stimulating hormone-induced adenylyl cyclase activity and proliferation in immature porcine Sertoli cell via sst2 receptor

The potential involvement of somatostatin (SRIF) in testicular function was studied by using as a model primary cultures of purified immature porcine Sertoli cells. In the present report we show that Sertoli cells express mRNA for sst2 SRIF receptor and display SRIF-sensitive adenylyl cyclase. Sensitivity of adenylyl cyclase to SRIF and its analogues is compatible with the pharmacological profile of this receptor type. Relevant cAMP production is similarly inhibited by SRIF in both basal and stimulated (by gonadotropin FSH or by forskolin) conditions. Moreover, the observed SRIF actions on Sertoli cells require functional coupling of specific membrane receptors to adenylyl cyclase via Gi proteins because pertussis toxin prevents SRIF-dependent inhibition of adenylyl cyclase in either basal or FSH-stimulated conditions. Given the potent antiproliferative actions of SRIF in other cell types, we further assessed the possible SRIF-dependent modulation of [(3)H]thymidine incorporation by Sertoli cells. Our data point to SRIF-mediated inhibition of both basal and FSH-stimulated [(3)H]thymidine uptake. This inhibition of Sertoli cell proliferation is, at least in basal conditions, also blocked by pertussis toxin pretreatment. Altogether, these data suggest that SRIF may play a role as an (local) inhibitor of FSH actions in testicular development.     

Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

Two amino acids, located in transmembrane domains VI and VII, determine the selectivity of the peptide agonist SMS 201-995 for the SSTR2 somatostatin receptor.

Human somatostatin receptor subtypes (SSTR1-5) bind their natural ligands SRIF-14 and SRIF-28 with high affinity. By contrast, short synthetic SRIF analogues such as SMS 201-995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub-nanomolar affinity only for the receptor subtype SSTR2. To understand the molecular nature of selective peptide agonist binding to somatostatin receptors we have now, by site-directed mutagenesis, identified amino acids mediating SMS 201-995 specificity for SSTR2. Sequentially, amino acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201-995, were exchanged for the corresponding SSTR2 residues. After three consecutive steps, in which eight amino acids were exchanged, a SSTR1 mutant receptor with high affinity for SMS 201-995 was obtained. Receptor mutants with different combinations of these eight amino acids were then constructed. A single Ser305 to Phe mutation in TM VII increased the affinity of SSTR1 for SMS 201-995 nearly 100-fold. When this mutation was combined with an exchange of Gln291 to Asn in TM VI, almost full susceptibility to SMS 201-995 was obtained. Thus, it is concluded that the specificity of SMS 201-995 for SSTR2 is mainly defined by these two amino acids in transmembrane domains VI and VII. Using the conjugate gradient method we have, by analogy to the well established structure of bacteriorhodopsin, built a model for SRIF receptor-ligand interactions that explains the importance of Gln291 and Ser305 for the selectivity of agonists.     

Homologous and heterologous regulation of somatostatin receptor 2

We previously demonstrated that phosphorylation of somatostatin receptor 2A (sst2A) is rapidly increased in transfected cells both by agonist and by the protein kinase C (PKC) activator phorbol myristate acetate (PMA). Here, we investigate whether PKC-mediated receptor phosphorylation is involved in the homologous or heterologous regulation of endogenous sst2 receptors in AR42J pancreatic acinar cells upon stimulation by agonist or by cholecystokinin (CCK) or bombesin (BBS). Somatostatin, PMA, CCK, and BBS all increased sst2A receptor phosphorylation 5- to 10-fold within minutes. Somatostatin binding also caused rapid internalization of the ligand-receptor complex, and PMA, CCK, and BBS all stimulated this internalization further. Additionally, sst2 receptor-mediated inhibition of adenylyl cyclase was desensitized by all treatments. Somatostatin, as well as peptidic (SMS201-995) and nonpeptidic (L-779,976) sst2 receptor agonists increased the EC(50) for somatostatin inhibition 20-fold. In contrast, pretreatment with BBS, CCK, or PMA caused a modest 2-fold increase in the EC(50) for cyclase inhibition. Whereas the PKC inhibitor GF109203X abolished sst2A receptor phosphorylation by CCK, BBS, and PMA, it did not alter the effect of somatostatin, demonstrating that these reactions were catalyzed by different kinases. Consistent with a functional role for PKC-mediated receptor phosphorylation, GF109203X prevented PMA stimulation of sst2 receptor internalization. Surprisingly, however, GF109203X did not inhibit BBS and CCK stimulation of sst2A receptor endocytosis. These results demonstrate that homologous and heterologous hormones induce sst2A receptor phosphorylation by PKC-independent and -dependent mechanisms, respectively, and produce distinct effects on receptor signaling and internalization. In addition, the heterologous hormones also modulate sst2 receptor internalization by a novel mechanism that is independent of receptor phosphorylation.     

The adenylyl cyclase family

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase.While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.     

Stimulation of the type III olfactory adenylyl cyclase by calcium and calmodulin.

Characterization of adenylyl cyclases has been facilitated by the isolation of cDNA clones for distinct adenylyl cyclases including the type I and type III enzymes. Expression of type I adenylyl cyclase activity in animal cells has established that this enzyme is stimulated by calmodulin and Ca2+. Type III adenylyl cyclase is enriched in olfactory neurons and is regulated by stimulatory G proteins. The sensitivity of the type III adenylyl cyclase to Ca2+ and calmodulin has not been reported. In this study, type III adenylyl cyclase was expressed in human kidney 293 cells to determine if the enzyme is stimulated by Ca2+ and calmodulin. The type III enzyme was not stimulated by Ca2+ and calmodulin in the absence of other effectors. It was, however, stimulated by Ca2+ through calmodulin when the enzyme was concomitantly activated by either GppNHp or forskolin. The concentrations of free Ca2+ for half-maximal stimulation of type I and type III adenylyl cyclases were 0.05 and 5.0 microM Ca2+, respectively. These data suggest that the type III adenylyl cyclase is stimulated by Ca2+ when the enzyme is activated by G-protein-coupled receptors and that increases in free Ca2+ accompanying receptor activation may amplify the primary cyclic AMP signal.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-adrenergic receptor: a potential mechanism of G protein activation

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.     

Insulin desensitized beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase in SK-N-MC cells.

Receptor cross-talk is an emerging field which investigates cross-regulation between distinct classes of receptors. In the present work, we investigated the influence of activating the insulin receptor, a tyrosine kinase receptor, on beta-agonist activation of adenylyl cyclase, which is mediated by a G protein-linked receptor. Treatment of SK-N-MC neuroepithelioma cells with insulin generated a marked attenuation of beta 1-adrenergic receptor-mediated stimulation of adenylyl cyclase. This effect required nanomolar concentrations of insulin, occurred within minutes of exposure of these cells to insulin, and did not result from down-regulation of beta-adrenergic receptors. Insulin alone reduced the maximal isoproterenol-mediated stimulation of adenylyl cyclase by 50%, while the co-addition of the phosphatase inhibitor sodium vanadate increased the magnitude of insulin inhibition to 90%. Insulin provides an additional avenue for heterologous desensitization of beta-adrenergic receptors and their transmembranal signalling pathway.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

Divergent effects of cations on follicle-stimulating hormone- and forskolin-activated adenylyl cyclase in granulosa cells

The divalent cations magnesium, calcium and manganese, and the monovalent cation, potassium, but not sodium, enhance binding of [125I]iodo-porcine follicle-stimulating hormone to follicle-stimulating hormone (FSH) receptors in membranes of porcine granulosa cells via an increase in the apparent number of binding sites. The objective of the present studies was to determine if increased binding of FSH to its receptor causes increased adenylyl cyclase activity in response to FSH, or conversely, if enhancement of the cyclase or one of its components causes increased binding, or if the two processes are modulated independently. MgCl2 and CaCl2, which both enhance binding in intact cells and in cell-free membrane preparations, had opposite effects on cyclase-MgCl2 stimulatory, CaCl2 inhibitory. In isotonic NaCl, MgCl2 did not enhance binding, but it did increase FSH-stimulated production of cyclic adenosine 3',5'-monophosphate (cAMP). NaCl did not enhance FSH binding and it did not enhance cyclase in cell-free membranes, but it did increase FSH- and forskolin-stimulated cAMP production in intact cells. In intact cells, maximally effective concentrations of MgCl2 and KCl were additive in enhancing cAMP production whereas the effects of NaCl and KCl together were synergistic. The results indicate that although cationic effects on FSH binding are not causally related to effects on cyclase, the cationic microenvironment of the granulosa cell membrane is critical to both FSH receptor and adenylyl cyclase functions.     

Hormone-sensitive adenylyl cyclase in preadipocytes cultured from adipose tissue: comparison with 3T3-L1 cells and adipocytes

The adenylyl cyclase system of preadipocytes derived from the stromal vascular fraction of perirenal rat fat pads was characterized. Unlike mature adipocytes, preadipocyte adenylyl cyclase was only weakly stimulated by catecholamines and adrenocorticotrophic hormone, but was stimulated by guanine nucleotides. Parathyroid hormone and 2-chloroadenosine also stimulated preadipocyte adenylyl cyclase. The adenylyl cyclase system of preadipocytes resembled that of undifferentiated 3T3-L1 cells. However, agents which induced the differentiation of the 3T3-L1 cell adenylyl cyclase system did not have a similar effect on preadipocytes. A medium (CDM6) which induced some differentiation of preadipocyte adenylyl cyclase was developed. The observations that the adenylyl cyclase system of preadipocytes and undifferentiated 3T3-L1 cells are similar, that preadipocyte adenylyl cyclase can be induced to develop along lines similar to early differentiation of 3T3-L1 cells, and that the adenylyl cyclase system of fully-differentiated 3T3-L1 cells has characteristics intermediate between preadipocytes and adipocytes, suggest that the differentiation of preadipocyte and 3T3-L1 adenyly cyclase in vitro mimics adipose adenylyl cyclase development in vivo. The increased catecholamine and ACTH stimulation, and reduced GTP and adenosine sensitivities of adipocytes compared to preadipocytes suggest that a number of genes affecting adenylyl cyclase-associated regulatory and receptor proteins are coordinately repressed and derepressed during development.