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-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.     

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.     

Alteration of somatostatin but not growth hormone-releasing factor pituitary binding sites in obese Zucker rats.

The present study was designed to determine whether the diminution of growth hormone (GH) secretion that occurs in obese Zucker rats is related to alterations of GH-releasing factor (GRF) or somatostatin (SRIF) pituitary binding sites. Cold saturation studies were performed in pituitary homogenates of 4-month-old lean and obese rats, using [125I-Tyr10]hGRF(1-44)NH2 as radioligand and [127I-Tyr10]hGRF-(1-44)NH2 as competitor, and in pituitary membrane preparations, using [125I-Tyr0, D-Trp8]SRIF14 as radioligand and [127I-Tyr0, D-Trp8]SRIF14 as competitor. In lean rats, analysis of the curves by the Ligand program revealed the presence of two distinct classes of GRF binding sites, the first being of high affinity (0.74 +/- 0.11 nM) and low capacity (118 +/- 31 fmol/mg protein), the second being of lower affinity (880 +/- 240 nM) and higher capacity (140 +/- 35 pmol/mg protein), and of a single class of SRIF binding sites (affinity: 0.40 +/- 0.12 nM; capacity: 24 +/- 6 fmol/mg protein). In obese rats, no difference was observed in GRF binding parameters for both classes of sites, but the concentration of somatostatin binding sites was reduced by 67% when compared to their lean littermates. These findings suggest that the SRIF pituitary receptors are down-regulated in obese Zucker rats and indicate that no alteration of GRF pituitary binding sites contribute to the blunted GH secretion observed in this model of obesity.     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Evidence for a single class of somatostatin receptors in ground squirrel cerebral cortex

In the present study we characterized high-affinity somatostatin (SRIF) binding sites (Kd = 2.06 +/- 0.32 nM and Bmax = 295 +/- 28 fmol/mg protein) in cerebral cortex membrane preparations of European ground squirrel using 125I-[Tyr0-D-Trp8]-SRIF14 as a radioligand. The inhibition of radioligand specific binding by SRIF14, as well as by its agonists (SRIF28, Tyr0-D-Trp8-SRIF14, SMS 201 995) was complete and monophasic, thus revealing a single population of somatostatinergic binding sites. Radioautographic analysis of 125I-[Tyr0-D-Trp8]-SRIF14 labeled brain sections confirmed the results of our biochemical study. The homogeneity of SRIF binding sites in the ground squirrel neocortex was not dependent on the animal's life-cycle phase.     

Distinct subsets of somatostatin receptors on cultured human lymphocytes

Somatostatin (SOM) is a neuroendocrine tetradecapeptide that suppresses specific functions of differentiated T-cells and antibody-producing cells. The Jurkat line of human leukemic T-cells and U266 IgE-producing human myeloma cells bound [I-Tyr11]SOM specifically. The maximal level of specific binding was attained by 1-2 h at 22 degrees C for both types of cells and reversed by 70-85% within 2-3 h after the addition of excess nonradioactive SOM. Computer-assisted Scatchard analysis of the competition curves revealed two classes of binding sites for both cells. An average of 144 and 1295 high affinity receptors per Jurkat and U266 cells had a Kd value of 3 pM and 5 pM, respectively, whereas a large number of low affinity sites had Kd values of 66 nM and 100 nM. The affinity of the analogs somatostatin 28, [I-Tyr11]SOM, and [D-Trp8, D-Cys14]SOM for Jurkat and U266 cell lines, relative to SOM, suggested a degree of specificity similar to receptors on neuroendocrine cells.     

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein.

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 microM 5'-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 +/- 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 +/- 0.7 nM) to one site of intermediate affinity (KD = 0.52 +/- 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts.

Somatostatin (somatotropin release inhibiting factor; SRIF) has widespread functions as a modulator of neural activity as well as of endocrine and exocrine secretion. In the present paper, the binding characteristics of somatostatin receptors have been investigated in rat long bones using the stable analogue, 125I-SDZ 204-090, as a ligand. Binding studies revealed the presence of a single class of high-affinity binding sites for 125I-SDZ 204-090 on cells prepared from neonatal rat long bones with an equilibrium dissociation constant (KD) of 70.1 +/- 8.2 pM (n = 3). An excellent correlation was found between the ability of various somatostatin analogues to inhibit growth hormone in pituitary cells and to displace the binding of 125I-SDZ 204-090 to the bone cell preparation, indicating that the receptors are very similar, if not identical. The localization of the somatostatin-binding sites was examined by autoradiography after labelling in vitro and in vivo. The binding sites were shown by both procedures to be selectively localized to the metaphysis of rat long bones. The labelling experiments in vivo indicate that these receptors can be reached in the living animal by circulating somatostatin analogues. In addition, the analogue SMS 201-995 inhibited the forskolin-stimulated adenylate cyclase activity in bone cell suspensions. These results suggest that somatostatin could be an important regulatory factor in bone metabolism.     

Expression of nanomolar-affinity binding sites for melatonin in Syrian hamster RPMI 1846 melanoma cells.

Pharmacological studies indicate that Syrian hamster melanoma (RPMI 1846) cells possess a melatonin binding site similar to that found in normal hamster cells. A high correlation was observed for a series of compounds between the Ki in hamster hypothalamic membranes vs. RPMI 1846 membranes (r = 0.94, slope = 0.93, P less than 0.01, n = 14). Scatchard analysis of saturation binding of 2-[125I]-iodomelatonin to membranes (at 0 degrees C) indicated: Kd = 0.89 +/- 0.08 nM, Bmax = 6.2 +/- 2.9 fmol/mg protein (n = 3). Melatonin did not alter basal or forskolin-stimulated adenylate cyclase activity in RPMI 1846 membranes or intact cells. Therefore, in contrast to the picomolar-affinity receptor for melatonin in the mammalian hypothalamus and pars tuberalis, the putative nanomolar-affinity receptor is not coupled to adenylate cyclase. The RPMI 1846 cell line provides a useful model system for further studies of signal transduction via the nanomolar-affinity site for melatonin.     

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.     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

The Cholinergic Regulation of Intracellular Calcium in the Human Neuroblastoma, SH-SY5Y

The regulation of intracellular calcium by cholinergic agonists was investigated in the human neuroblastoma SH-SY5Y, loaded with fura-2. The resting free Ca2+ concentration in this cell line was 199 +/- 14 nM (mean +/- SEM, n = 19). At 1 mM extracellular Ca2+, high concentrations of carbachol and acetylcholine evoked a biphasic change in intracellular Ca2+ concentration, consisting of a transient initial peak followed by a decline to a plateau that was significantly higher than the basal level. Carbachol (0.5 mM) and acetylcholine (10 microM) caused a maximal increase in the intracellular Ca2+ concentration, reaching a peak of 465 +/- 52 (mean +/- SEM, n = 12) and 422 +/- 48 nM (mean +/- SEM, n = 7), respectively, in less than 4 s. This initial calcium transient declined to a plateau of 268 +/- 36 and 240 +/- 27 nM for carbachol and acetylcholine, respectively, in approximately 40 s. The plateau persisted until the agonist was displaced by the addition of antagonist. Atropine, hexahydrosiladifenidol (HHSD), pirenzepine, and methoctramine inhibited the carbachol-evoked initial calcium transient with Ki values of 0.85 +/- 0.05, 8.3 +/- 1.6, 411 +/- 36, and 240 +/- 46 nM (mean +/- SEM, n = 3), respectively, and the acetylcholine-induced initial calcium transient with Ki values of 0.48 +/- 0.18, 13.5 +/- 8.5, 192 +/- 32, and 414 +/- 25 nM (mean +/- SEM of two experiments), respectively, results suggesting that an M3 muscarinic receptor was predominantly mediating these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical characterisation of somatostatin in ruminants

Following development and validation of a radioimmunoassay for somatostatin, the immunoreactivity of this peptide in the plasma of ruminants was measured and the levels in sheep were 9-31 pM (mean 18 +/- 7 pM, n = 48), in lambs 10-54 pM (mean 25 +/- 10 pM, n = 18) and in calves 5-35 pM (mean 12 +/- 6 pM, n = 22). Somatostatin-like immunoreactivity was present in sheep in high concentrations in the antrum (2342 +/- 280 pmol/g wet weight), duodenum (446 +/- 73 pmol/g) and pancreas (832 +/- 208 pmol/g). Lower concentrations (6-150 pmol/g) were found in other regions of the gastrointestinal tract. Molecular sieve chromatography on Bio-Gel P-10 showed that while most of the somatostatin in the antrum was somatostatin-14, in the duodenum about 30% of the total immunoreactivity was somatostatin-28.     

Altered release of growth hormone from dispersed adenohypophysial cells of streptozotocin diabetic rats. I. Effects of growth hormone releasing factor and somatostatin

As growth hormone has been implicated in the "dawn phenomenon," an early morning rise in serum glucose, we have studied the control of growth hormone release in diabetes using an acutely dispersed system of adenohypophysial cells from normal or diabetic rats (65 mg/kg streptozotocin, 8 days before sacrifice; serum glucose, 490 +/- 17 mg/dL). Growth hormone release is normally controlled by the two hypothalamic hormones, growth hormone releasing factor and somatostatin. We have found cells of the diabetic rats exhibit changes in sensitivity that result in increased growth hormone release in static incubation. In normal cells, rat growth hormone releasing factor increases growth hormone release three- to four-fold with an EC50 of 151 +/- 27 pM (n = 7). In contrast, in cells from diabetic rats, there was a significant (twofold) increase in sensitivity to growth hormone releasing factor (EC50 = 75 +/- 15 pM, n = 7) which resulted in increased growth hormone release with lower but not maximal (10 nM) growth hormone releasing factor. Basal nonstimulated release was unchanged. Somatostatin inhibition of stimulated growth hormone release was reduced (n = 7); half-maximal inhibition occurred with 0.21 +/- 0.03 nM (normal) and 0.76 +/- 0.17 nM somatostatin (diabetic). In perifusion the peak secretion rate was significantly lower for diabetic cells stimulated by a maximal dose of growth hormone releasing factor. These studies suggest somatotrophs of diabetic rats have altered sensitivity in vitro to the controlling hormones growth hormone releasing factor and somatostatin.     

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.     

Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ?[125I]ACTH-(13-24)?, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

High affinity binding of [125I-Tyr11]somatostatin-14 to human small cell lung carcinoma (NCI-H69)

The in vitro binding of [125I-Tyr11]somatostatin-14 (SRIF-14) to membranes prepared from cultured human small cell lung carcinoma (SCLC) cells (NCI-H69) has been characterized. Binding to SCLC was monophasic and of high affinity (Kd = 0.59 +/- 0.02 nM, n = 3). The estimated Bmax was 173 +/- 2.4 fmol/mg protein. Receptors were also present on solid NCI-H69 tumors grown in vivo in the athymic nude mouse. However, the concentration was only about 10% of that observed in cell culture. Biologically-active SRIF analogues were potent inhibitors of [125I-Tyr11]SRIF-14 binding, and an analysis of the pharmacological specificity indicated that the SCLC receptor was of the peripheral (e.g., non-neural) subtype. The presence of SRIF receptors on SCLC membranes may indicate that SRIF has a role in regulation of SCLC function.