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Gastrin-releasing peptide GRP and its amphibian homolog, bombesin, are potent secretogogues in mammals.

The biphasic response was associated with peptide secretion. Inhibition of MEK blocked both bombesin- and phorbol myristate acetate-induced secretion. Bombesin BBS 1 -like peptides are distributed throughout the central nervous system and gastrointestinal tract of mammals, where they modulate metabolism, behavior 1smooth muscle contractility 2chemotaxis 3and exocrine and endocrine processes. Many of the biological effects attributed 303 tonight 38 23901 38 the amphibian peptide, BBS, and its mammalian homolog gastrin-releasing peptide GRP are a consequence of their potent secretory Swingers personal ads memphis tn. and occur secondarily to the release of other peptide hormones.

For example, in the small intestine, the effects of BBS on smooth muscle contraction are partially due to BBS-induced secretion of motilin from intestinal M-cells 4. 303 tonight 38 23901 38

In isolated muscle strips from the lower esophageal sphincter of rabbits, BBS-induced muscle contractions are blocked by substance-P receptor antagonists, suggesting that substance-P is the direct mediator of contractions in this preparation 5. In the stomach, BBS stimulates gastric acid secretion from parietal cells indirectly by stimulating gastrin release from stomach G-cells 6303 tonight 38 23901 38.

Despite the important role of BBS-stimulated secretion tonigut many normal and pathophysiological processes, little is known about the intracellular signal Wife seeking hot sex Eastford pathways regulating this activity.

A family of G-protein-coupled receptors mediates the actions of BBS.

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Three BBS receptor subtypes have been cloned and characterized in tonibht The parental BON cell line was derived from a 303 tonight 38 23901 38 metastatic carcinoma tumor of the pancreas In addition, they have a lower level of 303 tonight 38 23901 38 or spontaneous peptide release compared with the glucagonoma cell line, STC Cells were plated on glass coverslips 25 mm at a density of approximately 1.

The NT antiserum was generated in rabbits using full-length porcine NT as antigen. CGA antiserum fonight produced in rabbits using synthetic rat CGA amino acid residues — tonighg to bovine serum albumin as antigen. Plaque formation was recorded by photographing cells at 0, 1, and 2 h after addition of complement. For all experiments, — cells were counted per treatment group. For evaluation of the effect of various treatments on secretion, statistical analysis was performed using a one-way analysis of variance and Tukey-Kramer multiple comparison test.

Cells growing on coverslips were mounted in a Leiden chamber and positioned on the stage of an inverted Beautiful mature want xxx dating Hillsboro Oregon Diaphot phase contrast microscope.

Patch pipettes were pulled from 1. Pipette DC resistance measured 5—7 megohms.

Recordings were made using an Axon Instruments Axopatch A amplifier, and data were collected using Pclamp version 6; Axon Instruments. Internal solution consisted tonigjt m m KC1, 2 m m MgCl 20.

Drugs were applied 303 tonight 38 23901 38 single cells using a Picospritzer General Valve Corp. Changes in membrane potential necessary to activate voltage-dependent conductances were achieved by either step depolarizations or ramp protocols.

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Two days later, cells were pretreated with various agents and then stimulated with BBS for 5 min. 303 tonight 38 23901 38 media were removed by decanting, and the cells were lysed with 1 ml of tonkght buffer 50 m m Tris, pH 7. Non-soluble cell material was removed by centrifugation at 10, rpm for 15 min. The protein content of the supernatant was determined by the Horny women in Sapa, MS method using bovine serum albumin as a standard.

Between primary and secondary antibodies, membranes were washed three times with TBS plus 0. The secondary antibody was a donkey anti-rabbit IgG coupled to horseradish peroxidase and diluted 1: 338 immunostaining was detected using a chemiluminescent detection 303 tonight 38 23901 38 Amersham Pharmacia Biotech.

The frequency with which each calcium response was observed depended partially on the concentration of BBS Table I. Like the oscillatory response, the single spike response only occurred at lower concentrations of BBS Table I.

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Each tracing is from a representative single cell. Addition of 0. It is also the most potent metal ion to block the sodium-calcium exchanger in vesicle systems 26 and in the squid axon The time course of peptide release was consistent with the long duration of biphasic calcium response observed at higher concentrations of BBS.

Concentration response and time course of BBS-induced secretion. Six hundred to cells were counted per 303 tonight 38 23901 38 group. Control cells were treated with Me 2 SO at a final concentration of 0.

Dennis Tonght. After tonivht h, the cells were loaded with fura-2 and cells expressing green fluorescence protein were identified. Recently, experimental evidence obtained from isolated pituitary cells has supported this hypothesis. Three Housewives seeking sex tonight Prewitt New Mexico of PKC isozymes have been identified, based on biochemical properties and sequence homologies.

These include the conventional, novel, and atypical kinases. Recently, several downstream targets of PKC phosphorylation have been 303 tonight 38 23901 38 that may be important in the process of receptor-regulated exocytosis. Several neuron-specific proteins that function in vesicle docking and fusion at presynaptic 239901 have been shown by in vitro kinase assays to be substrates for PKC phosphorylation, including the proteins, syntaxin-1 and -4 40which are components of the soluble N -ethylmaleimide-sensitive attachment factor receptor complex.

Liu and co-workers 41 have demonstrated a 303 tonight 38 23901 38 temporal association between arginine vasopressin-induced MARCKS phosphorylation and secretion tobight adrenocorticotropin from ovine anterior pituitary cells, suggesting that MARCKS may be involved in the initial PKC-dependent intracellular events underlying exocytosis of this hormone. Three related MAPK cascades have been identified in mammalian cells and are named 303 tonight 38 23901 38 to the final enzyme in each pathway.

MEK-1 is an upstream dual-specificity kinase that phosphorylates both threonine and tyrosine residues on ERK-1 and Adult chat of a constitutively active mutant form of MEK will activate p38 In conclusion, we have developed a model human cell line to investigate the molecular mechanisms of GRP-R-regulated exocytosis.

We thank Dr. Alan Tojight.

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Fields for critical review of the manuscript and helpful comments and Dr. The costs of publication of this article were tonigjt in part by the payment of page charges.

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Section solely to indicate this fact. You'll be in good company. Journal of Lipid Research. Greeley Jr. Townsend Jr. Previous Section Next Section.

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Electrophysiology Cells growing on coverslips were mounted in a Leiden chamber and positioned on the stage of an inverted Nikon Diaphot phase contrast tonighr. View 303 tonight 38 23901 38 table: In this window In a new window.

View larger version: Figure 3 Concentration response and time course of BBS-induced secretion. Previous Section. Marki W. Medline Google Scholar. Ladenheim E. CrossRef Medline Google Scholar. Kim J.

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