Release Of Bombesin-like Immunoreactivity Research Articles

NO releases bombesin-like immunoreactivity from enteric synaptosomes by cross-activation of protein kinase A.

The effect of nitric oxide (NO) on the release of bombesin-like immunoreactivity (BLI) was examined in synaptosomes of rat small intestine. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10(-7) to 10(-4) M) significantly stimulated BLI release. In the presence of the NO scavenger oxyhemoglobin (10(-3) M) or the guanylate cyclase inhibitor ODQ (10(-5) M), SNAP-induced BLI release was antagonized. In addition, SNAP increased the synaptosomal cGMP content and elevation of cGMP levels by zaprinast (3 x 10(-5) M), an inhibitor of the cGMP-specific phosphodiesterase (PDE) type 5, and increased basal and SNAP-induced BLI release. NO-induced BLI release was blocked by Rp-adenosine 3',5'-cyclic monophosphorothioate (3 x 10(-5) M and 10(-4) M), an inhibitor of the cAMP-dependent protein kinase A, whereas KT-5823 (3 x 10(-6) M) and Rp-8-(4-chlorophenylthio)-cGMP (5 x 10(-5) M), inhibitors of the cGMP-dependent protein kinase G, had no effect. Because cGMP inhibits the cAMP-specific PDE3, thereby increasing cAMP levels, the role of PDE3 was investigated. Trequinsin (10(-8) M), a specific blocker of PDE3, stimulated basal BLI release but had no additive effect on NO-induced release, suggesting a similar mechanism of action. These data demonstrate that because of a cross-activation of cAMP-dependent protein kinase A by endogenous cGMP BLI can be released by NO from enteric synaptosomes.

Effect of endogenous opioids on vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from the perfused rat stomach

The aim of the present study was to evaluate the effect of the opiate receptor antagonist naloxone on vagally stimulated secretion of bombesin-like immunoreactivity (BLI), somatostatin and gastrin from the isolated rat stomach, which was perfused via the celiac artery with Krebs-Ringer buffer. Vagal stimulation was performed for 10 min with 1 ms, 10 V and 2, 5, 10 or 20 Hz, respectively. In control experiments BLI release increased significantly above basal secretion during a stimulation frequency of 10 Hz (1367 ± 357 pg/10 min; P < 0.001) and 20 Hz (996 ± 202 pg/10 min; P < 0.01), but not at 2 and 5 Hz. In comparison to the controls naloxone (10 −6 M) significantly increased BLI secretion at 5 Hz by 573 ± 150 pg/10 min ( P < 0.05), but attenuated the BLI response to higher stimulation frequencies of 10 and 20 Hz to 284 ± 143 pg/10 min ( P < 0.001) and 490 ± 114 pg/10 min ( P < 0.01), respectively. At 2 Hz naloxone had no effect on BLI release. As shown previously the cholinergic blocker atropine (10 −7 M) induced a significant BLI release during vagal stimulation at 2 Hz (680 ± 233 pg/10 min; P < 0.01) and 5 Hz (935 ± 324 pg/10 min; P < 0.05), but was without effect at 10 and 20 Hz compared to the controls. The effects of the combination of naloxone and atropine were similar to naloxone and atropine alone. Naloxone had no effect on vagal or GRP-induced regulation of gastrin and somatostatin release. In conclusion, the present data demonstrate that endogenous opioids contribute to vagal regulation of BLI secretion. Stimulatory or inhibitory actions of endogenous opioids depend on the stimulation frequency employed. Further studies in isolated nerve endings are required to discriminate direct from indirect actions of endogenous opioids on the BLI neurons.

Release of bombesin-like immunoreactivity from synaptosomal membranes isolated from the rat ileum.

In the enteric nervous system, direct effects on peptidergic neurotransmitter release are difficult to assess since the neuronal network predisposes to numerous interactions between the various transmitter systems. The aim of the present study was to examine the release of bombesin-like immunoreactivity from isolated nerve synapses of the enteric nervous system. Enriched synaptosomal fractions were obtained by using homogenized tissue from rat ileum, which was subjected to various steps of differential and sucrose density centrifugation. Specific binding of [3H]saxitoxin served as a marker for neuronal membranes. For comparison, the content of bombesin-like immunoreactivity was determined. Both the enriched synaptosomal fraction (mitochondrial fraction II or P2) and the purified synaptosomal fraction (F2), obtained after discontinuous sucrose density centrifugation, showed substantial enrichment of the neuronal marker [3H]saxitoxin and bombesin-like immunoreactivity. The basal release of bombesin-like immunoreactivity was 52 +/- 17 pg/mg (100%). KCl-evoked depolarization (65 mM) significantly stimulated the release of bombesin-like immunoreactivity to 142.2% (P < 0.05, n = 17). The release was abolished in Ca(2+)-free medium. Stimulation of the release of bombesin-like immunoreactivity was also observed in the presence of the Ca2+ ionophore A-23187 (10(-6) M: 129%, P < 0.05, n = 17), supporting the role of Ca2+ in the release process. Cholinergic stimulation with carbachol elicited a significant dose-dependent release of bombesin-like immunoreactivity (10(-8) M: 106%, 10(-7) M: 175%, P < 0.05, 10(-6) M: 156%, P < 0.05, 10(-5) M: 115%, n = 14), which was reduced by atropine (10(-6) M: 99%, P < 0.01, n = 14). The basal value was 67 +/- 9 pg/mg (100%). The different effects of the muscarinic M1 receptor antagonist pirenzepine, which stimulated release of bombesin-like immunoreactivity in combination with carbachol 10(-6) M (10(-6) M: 123%, n = 10), and of the muscarinic M2 receptor antagonist AFDX 116, which attenuated release of bombesin-like immunoreactivity evoked by carbachol (10(-5) M: 66%, P < 0.01, 10(-6) M: 88%, n = 10), strongly suggest modulation of the release of bombesin-like immunoreactivity at the presynaptic receptor site through an excitatory muscarinic M2 receptor. The basal value was 46 +/- 9 pg/mg (100%). In summary, bombesin-like immunoreactivity can be released from these synaptosomes by both depolarization with KCl in a Ca(2+)-dependent manner and by cholinergic stimulation. The synaptosomes of intrinsic nerves of the gut offer an approach to study the release of neuropeptides and neurotransmitters at the subcellular level independent of the ganglionic network.

Bombesin-like peptides in alveolar macrophage: Increased release in pulmonary inflammation and fibrosis

Rat bronchoalveolar cells (99% alveolar macrophages (AM)) were obtained by bronchoalveolar lavage and examined for their content of bombesin-like immunoreactivity (BLI) by radioimmunoassay (RIA), immunocytochemistry and high performance liquid chromatography (HPLC) analysis. Rat AM contained and released in their culture media significant levels of BLI, the major molecular form corresponding to gastrin-releasing peptide (GRP). Release of BLI by AM was not affected by in vitro activation of AM with lipopolysaccharide and muramyl dipeptide, but was enhanced following in vivo treatment with inflammatory agents. AM from animals with inflammation and fibrosis released higher levels of BLI than controls at 3 and 6 weeks after treatment. These changes were correlated with a significant increase in the proportion of low density mature AM as determined by Percoll density gradient fractionation. Together, our data indicate that increased release of BLI by AM may be related to AM maturation and support a role for bombesin-like peptides as modulator(s) of inflammatory reactions.

Modulatory Effect of Insulin on Rat Small Intestinal Motility and Peptide Release in vitro

Intact segments of rat ileum were stimulated in vitro by either electrical field stimulation (EFS) or cholinergic stimulation with carbachol, in the presence of absence of varying insulin concentrations (50, 100 and 200 microU/ml), and the contraction in the longitudinal axis was recorded. Insulin had no significant influence on the carbachol contractile dose-response curve nor did it affect the cholinergically mediated 'on-contraction' at onset of the electrical stimulus. The 'off-contraction' occurring at cessation of the electrical stimulus was partly mediated by a cholinergic and partly by a noncholinergic and nonadrenergic mechanism, and decreased over time with repeated stimulation. This decay with time was significantly attenuated by insulin. In the presence of insulin, release of bombesin-like immunoreactivity induced by carbachol or EFS significantly increased. On the other hand, the release of somatostatin-like immunoreactivity was suppressed by 50-70% in the presence of insulin. These results demonstrate that in vitro insulin can modify both the motility responses of isolated segments of rat ileum and the neuropeptide release from such segments.

Vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from perfused rat stomach. Effect of stimulation frequency and cholinergic mechanisms

The isolated stomach of rats was vascularly perfused to measure the secretion of gastrin, somatostatin (SLI) and bombesin-like immunoreactivity (BLI). The gastric lumen was perfused with saline pH 7 or pH 2, and electrical vagal stimulation was performed with 1 ms, 10 V and 2, 5 or 10 Hz, respectively. Atropine was added in concentrations of 10 −9 or 10 −7 M to evaluate the role of cholinergic mechanisms. In control experiments, vagal stimulation during luminal pH 2 elicited a significant increase of BLI secretion only at 10 Hz but not at 2 and 5 Hz. Somatostatin release was inhibited independent of the stimulation frequency employed. Gastrin secretion at 2 Hz was twice the secretion rates observed at 5 and 10 Hz, respectively. At luminal pH 7 BLI rose significantly at 5 and 10 Hz. SLI secrtion was decreased by all frequencies. Gastrin secretion at 2 and 5 Hz was twice as high as during stimulation with 10 Hz. Atropine at doses of 10 −9, 10 −8, 10 −7 and 10 −6 M had no effect on basal secretion of BLI, SLI and gastrin. At luminal pH 2, atropine increased dose-dependently the BLI response at 2 and 5 but not at 10 Hz. The decrease of SLI during 2 and 5 Hz but not 10 Hz was abolished by atropine 10 −9 M. SLI was reversed to stimulation during atropine 10 −7 M at all frequencies. The rise of gastrin at 2 Hz was reduced by 50%. At luminal pH 7, atropine had comparable effects with a few differences: the BLI response at 10 Hz was augmented and the gastrin response to 2 and 5 Hz was reduced. In conclusion the present data demonstrate a frequency and pH-dependent stimulation of BLI and gastrin release. The stimulation of BLI is predominantly due to atropine-insensitive mechanisms while muscarinic cholinergic mechanisms exert an inhibitory effect on BLI release during lower stimulation frequencies (2 and 5 Hz) independent of the intragastric pH and also during higher frequencies at neutral pH. Both, atropine sensitive and insensitive mechanisms are activated frequency dependent. The atropine-sensitive cholinergic mechanisms but not the noncholinergic mechanisms involved in regulation of G-cell function are pH and frequency dependent. Somatostatin is regulated largely independent of stimulation frequency and pH by at least two pathways involving cholinergic mechanisms of different sensitivity to atropine. These data suggest a highly differentiated regulation of BLI, gastrin and SLI secretion and the interaction between these systems awaits further elucidation.

Neuroendocrine responses to stimulation of the splanchnic nerves in bursts in conscious, adrenalectomized, weaned lambs.

1. Effects of stimulation of the peripheral ends of the splanchnic nerves below behavioural threshold at either 4 or 7 Hz continuously for 10 min, or at 40 or 70 Hz for 1 s at 10 s intervals for 10 min. have been compared in conscious adrenalectomized lambs. 2. Both patterns of stimulation resulted in an abrupt rise in mean aortic blood pressure of closely similar extent which was associated with reflex bradycardia. 3. At the lower frequencies both patterns of stimulation elicited a closely similar rise in mean plasma glucose, glucagon and pancreatic polypeptide concentration, but the fall in mean plasma insulin concentration was significantly greater during continuous stimulation. 4. Unlike other species in which the release of NPY and bombesin-like immunoreactivity (BLI) is potentiated by intermittent high-frequency stimulation, no significant differences were produced by changing the pattern of stimulation. The release of BLI was found to be frequency related over the ranges tested (4-7 Hz continuously and 40-70 Hz in bursts) whereas the release of NPY was not. 5. Splanchnic nerve stimulation also produced detectable rises in the mean plasma concentrations of noradrenaline and adrenaline. The mean average concentration of noradrenaline during stimulation in bursts was significantly higher than that during continuous stimulation (P less than 0.02). There was also a steady rise in mean plasma 3,4-dihydroxyphenylacetic acid (DOPAC) during stimulation followed by a further rise to significantly higher values (P less than 0.02) following stimulation in bursts at 40 Hz. 6. It is concluded that the pattern of stimulation is a less important determinant of autonomic responses to splanchnic nerve stimulation in sheep than in certain other species.

Structural identification, subcellular localization and secretion of bovine adrenomedullary neuromedin C [GRP-(18–27)

Bombesin-like immunoreactivity (BLI) was purified from acid (HCl) extracts of bovine adrenal medulla. High performance liquid chromatography (HPLC) on a μ-Bondapak C18 column revealed the presence of five molecular forms of BLI, one coeluting with synthetic gastrin releasing peptide (GRP), the mammalian counterpart of amphibian bombesin, one coeluting with neuromedin C, one coeluting with neuromedin B and the two other ones coeluting with the oxidized forms of neuromedins B and C. The material corresponding to neuromedin C was purified to homogeneity and its amino acid composition and sequence corresponded to those expected for neuromedin C. HPLC analysis on an analytical SP-5PW column of subcellular extracts of bovine adrenal medulla indicated that neuromedin C is almost exclusively localized in secretory granules. The neuropeptide function of neuromedin C and/or other BLI peptides at this level was supported by the stimulatory effect of carbamylcholine (500 μM) on the release of BLI (4.5-fold increase over the basal release of 19 fmol/5 min) from perfused bovine adrenal glands.

Release of bombesin-like immunoreactivity from the isolated perfused rat stomach

Release of bombesin-like immunoreactivity from the isolated perfused rat stomach

The use of a rat isolated ileal preparation to investigate the release of neurotensin.

An isolated and perfused preparation of rat ileum was used to investigate the effects of cholinergic, adrenergic and bombesin stimuli on neurotensin release into the vascular compartment. A vigorous release of neurotensin like immunoreactivity (NTLI) to 200% above basal values in response to intraluminal infusions of emulsified soybean oil (Intralipid) demonstrated the physiological responsiveness of the preparation. Carbachol significantly stimulated the release of both NTLI and bombesin like immunoreactivity (BLI), with maximal responses at 5 X 10(-9) mol/l carbachol of 100% and 400% above basal values for NTLI and BLI, respectively. Noradrenaline at 10(-6) and 10(-4) mol/l caused no significant release of NTLI but markedly inhibited spontaneous BLI release. Synthetic amphibian bombesin caused a marked release of NTLI to 81% and 100% above basal at infusion concentrations of 5 X 10(-11) and 5 X 10(-10) mol/l respectively. These results suggest that there is either a direct effect of cholinergic agents on the N cells bringing about NTLI release, or an indirect effect via the release of bombesin like peptides from intrinsic gut neurones. This preparation provides a useful model to study the complex neural, paracrine and endocrine interactions in the gastrointestinal tract.

Characteristics of the neuroendocrine responses to stimulation of the splanchnic nerves in bursts in the conscious calf.

Neuroendocrine responses to splanchnic nerve stimulation in bursts (at 40 Hz for 1 s at 10 s intervals for 10 min) have been investigated in conscious adrenalectomized calves, 3-6 weeks after birth, in the presence of various pharmacological blocking agents. Preganglionic blockade with hexamethonium abolished all the neuroendocrine responses that were monitored. Pre-treatment with phentolamine significantly reduced, but failed to eliminate, the release of both pancreatic glucagon and pancreatic polypeptide. In the presence of phentolamine splanchnic nerve stimulation produced a massive rise in arterial plasma insulin concentration. None of these pancreatic neuroendocrine responses was significantly affected by additional pre-treatment with propranolol. The rise in mean plasma insulin concentration which occurred in calves pre-treated with both phentolamine and propranolol was significantly reduced by atropine. Release of bombesin-like immunoreactivity (BLI) was unaffected by total post-ganglionic adrenergic and cholinergic blockade. The results indicate that pancreatic endocrine responses to splanchnic nerve stimulation may be attributable, at least in part, to release of BLI in this species.

Neuroendocrine responses to stimulation of the splanchnic nerves in bursts in the conscious adrenalectomized calf.

Effects of stimulation of the peripheral ends of the splanchnic nerves below behavioural threshold at either 4 or 2 Hz continuously for 10 min, and at 40 or 20 Hz for 1 s at 10 s intervals for 10 min, have been compared in conscious calves. Cardiovascular responses were apparently unaffected by the pattern of the stimulus, whereas pancreatic neuroendocrine responses were significantly enhanced by stimulation in bursts, as was the rise in mean arterial plasma glucose concentration. Release of bombesin-like immunoreactivity was substantially potentiated by intermittent stimulation at relatively high frequencies and the significance of this discovery is discussed in relation to the effects that this peptide is known to evoke in this species.

Release of bombesin-like immunoreactivity from the isolated perfused rat stomach

In the present study the release of bombesin-like immunoreactivity (BLI), somatostatin and gastrin was determined form the isolated perfused rat stomach. Gastric inhibitory polypeptide (GIP, 2 X 10(-9) M) had no effect on BLI while stimulating somatostatin and gastrin release. In these experiments the luminal pH of the stomach was kept at pH 7. Reduction of the luminal pH to 2 resulted in an inhibition of BLI secretion by GIP while gastrin release was abolished and somatostatin remained unaffected compared to luminal pH 7. Acetylcholine (10(-6) and 2 X 10(-6) M) elicited a dose-dependent stimulation of BLI secretion while gastrin was stimulated and somatostatin secretion suppressed independent of the administered dose. The present data demonstrate that release of bombesin-like immunoreactivity can be modulated by intestinal hormones and neurotransmitters and is integrated into the complex system of gastrointestinal neuroendocrine regulation.