Bombesin receptor in membranes from Swiss 3T3 cells. Binding characteristics, affinity labelling and modulation by guanine nucleotides.

Bombesin-like neuropeptides, including mammalian gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells. In this study, we have characterized the bombesin receptor in membrane preparations from these cells. Addition of Mg2+ during cell homogenization was essential to preserve 125I-GRP binding activity in the resulting membrane preparation. The effect of Mg2+ was concentration dependent, with a maximum at 5 mM. Specific binding of 125I-GRP was saturable; Scatchard analysis indicated a single class of high-affinity sites of Kd = (2.1 +/- 0.3) x 10(-10) M at 15 degrees C and Kd = (1.9 +/- 0.4) x 10(-10) M at 37 degrees C, and a maximum binding capacity of 580 +/- 50 fmol/mg of protein (15 degrees C) or 604 +/- 40 fmol/mg of protein (37 degrees C). The kinetically derived dissociation constant was 1.5 x 10(-10) M. 125I-GRP binding was inhibited in a concentration-dependent manner by various peptides containing the highly conserved C-terminal heptapeptide of the bombesin family, including bombesin, GRP, neuromedin B and the 8-14 fragment of bombesin. In contrast, a variety of structurally unrelated mitogens and neuropeptides had no effect. The cross-linking agent ethyleneglycolbis(succinimidylsuccinate) covalently linked 125I-GRP to a single Mr 75 000-85 000 protein in membrane preparations of 3T3 cells. Affinity labelling of this molecule was specific and dependent on the presence of Mg2+ during membrane preparation. Finally, the non-hydrolysable GTP analogue guanosine-5'-[gamma-thio]triphosphate (GTP[S]) caused a concentration-dependent inhibition of 125I-GRP binding and cross-linking to 3T3 cell membranes [concentration giving half-maximal inhibition (IC50) approximately 0.2 microM]. The inhibitory effect was specific (GMP, ATP or ATP[S] had no effect at 10 microM) and was due to an increase in Kd from (1.7 +/- 0.2) x 10(-10) M to (4.3 +/- 0.6) x 10(-10) M in the presence of 10 microM-GTP[S]. This modulation of ligand affinity and cross-linking implies that the bombesin receptors that mediate mitogenesis in Swiss 3T3 cells are coupled to a guanine-nucleotide-binding-protein signal-transduction pathway.     

Interleukin 2 increases T lymphocyte membrane mobility before the rise in cytosolic calcium concentration

Using stopped-flow fluorometry with three different fluorescence probes [2-[(1-pyrenyl-butyryl)oxy]stearic acid, chlortetracycline and Quin 2], we have studied initial stage of T lymphocyte activation after interleukin 2 (IL-2) binding to a specific cell-surface receptor. After IL-2 binding to cytotoxic T lymphocyte (IL-2-dependent mouse LC7 and CTLL-2 cells), membrane mobilities of the cells increased first (4.5 +/- 0.3 s-1 for LC7 and 3.8 +/- 0.2 s-1 for CTLL-2), then calcium was released from intracellular stores into the cytoplasm (1.6 +/- 0.1 s-1 for LC7 and 2.1 +/- 0.1 s-1 for CTLL-2), and lastly, calcium was transported from the external medium into the cytoplasm (1.3 +/- 0.1 s-1 for LC7 and 1.5 +/- 0.1 s-1 for CTLL-2). The slowest process, the calcium influx from the external medium, was suppressed in the presence of a calcium channel blocking agent (verapamil). These observations give us a new information to discuss a model in T lymphocyte activation after IL-2 binding to cell-surface receptors.     

Chronic desensitization to bombesin by progressive down-regulation of bombesin receptors in Swiss 3T3 cells. Distinction from acute desensitization

Prolonged exposure (40 h) of Swiss 3T3 cells to bombesin induced homologous desensitization to bombesin and structurally related peptides including mammalian gastrin releasing peptide (GRP). The ability of bombesin to mobilize intracellular Ca2+, inhibit epidermal growth factor binding, and stimulate DNA synthesis was profoundly and selectively inhibited. In contrast, Ca2+ mobilization by either vasopressin or bradykinin was unaffected, indicating that chronic desensitization is mechanistically distinct from acute desensitization of Ca2+ mobilization. Prolonged (24 or 40 h) pretreatment with bombesin also induced a 78 +/- 5% loss of bombesin receptor binding sites in both intact and plasma membrane preparations of Swiss 3T3 cells without an apparent change in receptor affinity (Kd = 1.9 +/- 0.1 x 10(-9) M and Kd = 1.8 +/- 0.2 x 10(-9) M for control and pretreated cells, respectively). Loss of 125I-GRP binding was slow and progressive with half-maximal loss of binding occurring after 7 h and maximal after approximately 14 h. Cross-linking of 125I-GRP to intact cultures and membrane preparations revealed an identical time-dependent loss of the Mr = 75,000-85,000 cross-linked band, previously identified as the bombesin receptor. Prolonged exposure of the cells to phorbol 12,13-dibutyrate, epidermal growth factor, cholera toxin, or mitogenic combinations of these agents did not alter 125I-GRP binding. Receptor down-regulation and loss of mitogenic responsiveness to bombesin were: (a) induced in a parallel dose-dependent manner by bombesin (ED50 = 1 nM), GRP (ED50 = 2 nM), and neuromedin B (ED50 = 20 nM), but not by the biologically inactive fragment GRP (1-16); (b) inhibited by the specific bombesin antagonist [Leu13-psi(CH2NH)-Leu14] bombesin, and (c) reversed upon removal of bombesin with a similar time course (full recovery after 15 h). On the basis of these observations, we propose that prolonged pretreatment of Swiss 3T3 cells with bombesin induces homologous desensitization to peptides of the bombesin family by down-regulation of cell surface bombesin receptors.     

Somatostatin inhibition of phosphoinositides turnover in isolated rat acinar pancreatic cells: interaction with bombesin.

The effects of somatostatin-14 and bombesin on [3H]inositol phosphate accumulation were studied in 24 h myo-[3H]inositol-prelabeled cultured rat acinar cells. Bombesin, 10 nM, stimulated basal formation of phosphatidyl monophosphate (InsP1), phosphatidyl 4,5-biphosphate (InsP2) and inositol 1,4,5-triphosphate (InsP3) by 128 +/- 5.2%, 147 +/- 10% and 155 +/- 5%, respectively. At 5 s, the ED50 value for InsP3 stimulation was 0.70 +/- 0.2 nM. This stimulation was partly blocked (64 +/- 0.04% inhibition) by 10 ng/ml Bordetella pertussis toxin. In contrast to bombesin, somatostatin, 10 nM, inhibited basal InsP1, InsP2 and InsP3 formation. At 5 s, the inhibition degree for InsP3 was 18 +/- 2.5% and the IC50s values 1 +/- 0.09 nM, 1 +/- 0.12 nM and 0.07 +/- 0.005 nM for InsP1, InsP2 and InsP3, respectively. Bombesin-stimulated InsP3 formation was also inhibited by somatostatin. At 5 s, the inhibition degree was 85 +/- 3.5% at 10 nM and the IC50 value, 0.10 +/- 0.05 nM. Furthermore, somatostatin inhibition of bombesin stimulation was partly blocked (66 +/- 4% inhibition) by Bordetella pertussis toxin. These data therefore suggest that the acinar pancreatic cells contain a somatostatin receptor exerting a negative control on basal and bombesin receptor-stimulated phosphatidyl inositol turnover.     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes

The effect of phorbol esters on the proliferation and survival of interleukin-2(IL-2)-dependent cells was studied using an IL-2-dependent T cell line (CTLL-2) and blasts of BALB/c mouse spleen cells stimulated with Concanavalin A. Addition of phorbol 12,13-dibutyrate (PDBu) to CTLL-2 or ConA blasts induces a mitogenic response which is 25-40% of that elicited by IL-2. Interleukin 2 deprivation leads to a marked decline in the number of viable cells (50% of CTLL-2 cells have died after 8-10 hours incubation in IL-2-free medium). The mechanism of cell death seems to correspond to the suicide process known as apoptosis since an early degradation of DNA into oligonucleosome-size fragments could be observed after removal of the growth factor. When present, PDBu inhibits both the activation of the endonuclease and the development of the cell death process in CTLL-2 cells and ConA-blasts deprived of IL-2. Taken together, our results suggest that the tumor promoters phorbol esters inactivate in T cells the mechanism of cell elimination triggered by IL-2 deprivation and may help to explain why transformation of T cells decreases or even abolishes their requirements of IL-2 for survival and growth.     

The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C.

Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.     

Activation of the Na+/K+-ATPase by interleukin-2

Activated B61.SF.1 and CTLL-2 T lymphocyte clones which are strictly dependent on interleukin-2 (IL-2) for growth were used to study the activation of Na+/K+-ATPase. 50% of [3H]thymidine maximal incorporation was obtained when the extracellular concentration of Na+ or K+ was reduced to 50 or 2 mM, respectively. 'Quiescent' CTL clones stimulated with IL-2 showed an increase of 48-380% in ouabain-sensitive 86Rb uptake. Furthermore, this stimulation was completely inhibited by a monoclonal antibody PC.61 directed at the IL-2 receptor. The activation of the pump was dependent on the dose of IL-2, took place at the same doses of IL-2 that were required to stimulate cell proliferation and was linear for at least 30 min.     

Bombesin-like peptides stimulate gastrin release from isolated rat G-cells

Bombesin-like peptides as well as receptor-independent activators were tested for their effect on gastrin release from acutely dispersed rat gastric G-cells. The amphibian peptide bombesin as well as its mammalian analogues neuromedin B and neuromedin C stimulated gastrin release. Maximal responses were achieved with 10(-9) M bombesin (191.0 +/- 16.8% of basal release), 10(-8) M neuromedin C(205.9 +/- 17.6%) and 10(-7) M neuromedin B (162.2 +/- 10.4%), respectively. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and the synthetic diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) are receptor-independent activators of the protein kinase C. Both TPA (10(-6) M) and OAG (10(-5) M) stimulated gastrin release to 214.0 +/- 29.3% and 198.2 +/- 20.8% of basal, respectively. Calcium ionophore A23187 (10(-5) M) was the most effective stimulant tested (364.7 +/- 39.6%). Its effect was reversed by the calmodulin antagonist W 7 (10(-6)-10(-5) M). Finally, forskolin (10(-5) M), a direct activator of cAMP-formation, as well as the cAMP-analogue dbcAMP (10(-3) M) induced gastrin release. IN conclusion, neuromedin B is less potent and less effective than neuromedin C and bombesin in stimulating rat gastric G-cells. In addition, gastrin release is activated by calcium- and phospholipid-dependent as well as by cAMP-induced cellular signal transduction mechanisms.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dopamine Stimulates [3H]Phorbol 12,13-Dibutyrate Binding in Cultured Striatal Cells

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.     

Muscarinic stimulation of submucosal glands in swine trachea

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [3H]quinuclidinyl benzilate ([3H]QNB) and N-[3H]methylscopolamine ([3H]NMS) as ligands. Analysis of competitive displacement of ([3H]NMS binding by pirenzepine demonstrated the presence of M1- (27 +/- 2%) and M2G- (73 +/- 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by greater than 95%. After 7 days of DFP treatment, [3H]QNB binding to intact TSGC's decreased from 14.2 +/- 0.6 to 6.3 +/- 0.8 fmol/10(6) cells; similarly, [3H]NMS binding fell from 8.1 +/- 1.9 to 2.0 +/- 0.8 fmol/10(6) cells. The loss of mAChR's was predominantly of the M2G subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [3H]NMS. Mucus secretion was quantitated by measuring the release of 3H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 X 10(-5) M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such that only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro lipid metabolism in the rat pancreas. II. Effects of secretagogues on fatty acid metabolism, net lipolysis and ATP levels.

1. The concentration of carbamylcholine, bombesin, pancreozymin, pentagastrin and secretin evoking a similar 4--5-fold maximal increase in amylase secretion from rat pancreatic fragments were 3.10(-6), 10(-7), 10(-8), 3.10(-6), and 3.10(-6) M, respectively. The maximal concentration of vasoactive intestinal peptide tested (3.10(-6) M) increased amylase secretion by 250%. The six secretagogues could be separated into two groups according to their effects on lipid metabolism and ATP levels. 2. When used at their optimal concentrations, carbamylcholine, bombesin, pancreozymin, and pentagastrin lowered pancreatic ATP levels by 18-26% and increased net release of free fatty acids by 68-105%. 3. The effects of 3.10(-6) M carbamylcholine and 10(-8) M pancreozymin on the metabolism of 3H2O, D-[U-14C]glucose and [1-14C]acetate were similar; the incorporation of radioactivity in the fatty acid moiety of glycerolipids decreased by 20--50% whereas the incorporation of 3H from 3H2O and of 14C from [U-14C]glucose increased by 20--35% in the glycerol moiety. In addition, the oxidation of [U-14C]glucose, [1-14C]acetate and [1-14C]palmitate to 14CO2 increased by 15--32% while the esterification of [1-14C]palmitate, [1-14C]-linoleate, and [1-14C]arachidonate was inhibited by 14--23%. The spectrum of fatty acids labeled with [1-14C]acetate indicated an inhibition of the malonic acid pathway whereas the elongation of polyenoic fatty acids was unaltered.     

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Interleukin-1 suppresses mesangial cell growth via inhibition of Ca2+ entry

We have investigated the effect of interleukin-1 (IL-1) on the cell growth and Ca2+ homeostasis of rat mesangial cells in culture. DNA synthesis measured by [3H]thymidine uptake by mesangial cells was significantly inhibited by IL-1 (10 U/ml) and the calcium channel antagonist nicardipine (5 x 10(-6) M). 45Ca2+ uptake by mesangial cells was also significantly inhibited by IL-1 and nicardipine. The above observations support the premise that IL-1 suppresses the growth of mesangial cells via inhibition of extracellular Ca2+ entry to the cytosol.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Inhibition of bombesin-stimulated gastrin release from isolated canine G cells by bombesin antagonists

This study investigated the effects of two putative bombesin antagonists, [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P and [Leu13-psi-CH2NH-Leu14]bombesin, on bombesin-stimulated gastrin release from isolated canine G cells following short-term culture. Canine antral tissue was dispersed by sequential collagenase and EDTA treatment, and counterflow elutriation was used to enrich for G cells. Plates were seeded with 2 x 10(6) cells/mL in each well and cultured for 2 days prior to testing. Gastrin-containing and somatostatin-containing cells were identified by immunocytochemistry using the biotin-avidin-peroxidase method and accounted for 8.5 and 1%, respectively, of adhered cells. Basal gastrin secretion was 1.91 +/- 0.48% of total cell content. After a 2-h incubation period, bombesin (0.01-100 pM) stimulated gastrin release in a concentration-dependent fashion. The substance P analog, at a concentration of 1 microM, modestly inhibited bombesin-stimulated gastrin release from canine G cells. This analog also produced weak stimulation of basal gastrin release. In contrast, the bombesin analog, at a concentration of 1 microM, did not affect basal gastrin secretion. The bombesin analog completely blocked bombesin-stimulated gastrin release from 0.01 to 1 pM and produced greater than 50% inhibition at higher doses. The ability of the bombesin analog to directly inhibit bombesin-stimulated gastrin release from cultured canine G cells underscores its usefulness in studies involving the role of bombesin and its mammalian counterpart, gastrin-releasing peptide, in the control of gastrin cell function.     

Inhibitory effect of a long-acting somatostatin analogue on EGF-stimulated cell proliferation in Capan-2 cells.

Numerous studies have reported diverse effects of gut-derived regulatory peptides on growth of the normal pancreas, pancreatic neoplasms induced experimentally in animals, and pancreatic cancer cell lines, but the results of these investigations are rather controversial. The stimulatory effect of epidermal growth factor (EGF) on cell proliferation of pancreatic cell lines is well established. Whether this action can be modulated by somatostatin is not clear. Furthermore, it is not certain whether another regulatory peptide, cholecystokinin (CCK), affects the proliferation of these cells. In the present study we investigated the presence of CCK-A and CCK-B, as well as somatostatin-2 (SSTR2) receptors by RT-PCR, and studied the actions of EGF, CCK and octreotide on DNA synthesis in the human pancreatic adenocarcinoma cell line Capan-2. Octreotide, a long-acting somatostatin analogue was used as somatostatin agonist. Cells were cultured in RPMI-1640 medium. They were incubated in serum free medium containing 0.2% BSA in the absence (control) or the presence of the peptides. [3H]-thymidine incorporation into DNA was measured after 48 h of incubation. By means of RT-PCR analysis we were able to demonstrate SSTR2 expression, but not CCK-A or CCK-B receptor mRNA in Capan-2 cells. DNA synthesis evaluated by [3H]-thymidine incorporation was found to be increased by 45.2 +/- 5.6% in response to EGF (10(-8) M) and decreased by 11.7 +/- 2.6% to octreotide (10(-8) M) compared to controls (P < 0.01). The increase in [3H]-thymidine incorporation was significantly lower when EGF treatment was combined with octreotide administration (10.1 +/- 2.5% over control). In the concentration range of 10(-11)-10(-8) M, CCK did not alter significantly the incorporation of [3H]-thymidine into DNA in Capan-2 cells. In conclusion, these data support a role for EGF as a growth factor for the human pancreatic cancer cell Capan-2. Somatostatin may play an important role in regulating cell proliferation in Capan-2 cells both under basal, and growth factor-stimulated conditions. Our results suggest, however, that CCK receptors are not expressed, and CCK does not affect cell proliferation in this transformed pancreatic cell line.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.