Centrally administered bombesin modulates gastric motility

Intracerebroventricular bombesin alters arterial pressure and gastrointestinal transit in rats. In order to evaluate the influence of bombesin on arterial and gastric intraluminal pressure in a specific site in the central nervous system, we microinjected bombesin into the medial subnucleus of the nucleus tractus solitarius (mNTS) in 28 rats anesthetized with choralose. Bombesin (78 pmole in 25 nl), but not vehicle, caused an increase of tonic gastric intraluminal pressure (2.6 +/- 0.5 cm H2O) and of phasic gastric intraluminal pressures but did not acutely alter arterial pressure. The effect on tonic and phasic gastric intraluminal pressure was dose-dependent. The threshold dose was 7.8 pmole. Intravenous bombesin caused a similar dose-dependent rise in tonic gastric intraluminal pressure but did not significantly change the mean amplitude of phasic gastric intraluminal pressures. Transection of the cervical spinal cord and both cervical vagus nerves blocked the effect of centrally but not peripherally administered bombesin. We conclude that bombesin microinjected into the mNTS does not influence arterial pressure but does raise tonic and phasic gastric intraluminal pressures. Bombesin may act in the NTS as a central modulator of gastric motility.     

An inhibitory role of beta-endorphin in central cardiovascular regulation

The cardiovascular effects of beta-endorphin after administration directly into the nucleus tractus solitarii (NTS) of urethane anaesthetised rats were investigated. Unilateral injection resulted in a dose related fall in mean arterial pressure and heart rate. No change in respiratory frequency was prevented and the bradycardia reduced by pretreatment with locally applied naloxone (10 ng). This dose of the opiate antagonist had no effect on mean arterial pressure or heart rate when administered alone. Antiserum to beta-endorphin (1:50 dilution) caused a rise in pressure and a tendency towards tachycardia on injection into the NTS, while it completely blocked the depressor response and bradycardia induced by beta-endorphin. These results are consistent with the view that a beta-endorphin-like peptide has a depressor role in the central nervous system. The hypotension may result from an effect within the central connections of the baroreceptor reflex arc, probably at the level of the NTS.     

N-acetyl-GRP(20-26)-O-CH3 reverses intracisternal bombesin-induced inhibition of gastric acid secretion in rats

Intracisternal injection of 19 pmoles of bombesin in light-ether-anesthetized rats, five minutes after intracisternal vehicle, produced a 75% and 63% inhibition in gastric acid output and concentration, respectively, in 2-hour pylorus-ligated rats. Pretreatment of rats with the characterized peripheral bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 (1 nmole) reversed the inhibitory effect of bombesin on gastric acid output and concentration. In contrast, the related bombesin antagonist N-acetyl-GRP-O-CH2-CH3 (1 nmole) was ineffective in this system. In urethane-anesthetized, acute gastric fistula rats infused with pentagastrin, intracisternal N-acetyl-GRP(20-26)-O-CH3 protected against the inhibition in gastric acid output produced by intracisternal bombesin (19 pmoles). Thus the recently characterized peripheral bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 also appears to be effective in antagonizing central bombesin-induced inhibition in gastric acid secretion in two models. This represents a first report of a synthetic bombesin antagonist effective in reversing central bombesin-induced effects on gastric function.     

The roles of glucagon and adrenal epinephrine in mediating hyperglycemia induced by third cerebroventricular injection of bombesin

The roles of glucagon and adrenal epinephrine in mediating bombesin-induced central hyperglycemia were further studied in anesthetized rats. Bombesin (10(-9) mol) injected into the third cerebral ventricle produced an increase in plasma concentrations of glucose, glucagon, and epinephrine. Prior bilateral adrenalectomy completely prevented the hyperglucagonemic and hyperglycemic responses to third cerebral ventricle injection of bombesin. These results support the view that bombesin-induced increases in plasma glucose and glucagon are fully dependent on adrenal epinephrine secretion. Furthermore, during constant intravenous infusion of somatostatin, the hyperglycemic response to third cerebral ventricle injection of bombesin was not significantly influenced despite complete inhibition of the increase in plasma glucagon. Therefore, it is suggested that bombesin-induced central hyperglycemia is mainly mediated by epinephrine itself rather than via epinephrine-stimulated glucagon secretion.     

Insulin reduces the requirement for EGFR transactivation in bombesin-induced DNA synthesis

The binding of bombesin to its cognate G-protein coupled receptor stimulates quiescent Swiss 3T3 cells to re-initiate DNA synthesis and cell division. Addition of a non-mitogenic concentration of insulin dramatically potentiates bombesin-induced cell proliferation. We examined whether bombesin-induced EGFR transactivation mediates synergistic cell proliferation induced by bombesin and insulin. Treatment with selective EGFR tyrosine kinase inhibitors blocked EGFR transactivation, DNA synthesis, the transition of cells from quiescence into the cell cycle, and the expression of cyclins D1 and E induced by bombesin alone. In contrast, the inhibitors prevented cell cycle progression to a much lesser degree in cells stimulated with the combination of bombesin and insulin. Our results indicate that EGFR transactivation does not mediate synergistic cell proliferation induced by bombesin and insulin, and imply that insulin compensates for the requirement for EGFR transactivation in bombesin-induced DNA synthesis.     

Stimulation of K+ transport systems by Ha-ras

The expression of Ha-ras in quiescent NIH3T3 cells carrying a glucocorticoid-inducible human Ha-ras gene (Val-Gly mutation at codon 12) stimulates total 86Rb+ influx. This effect is predominantly due to an elevated 86Rb+ uptake through an ouabain-resistant, furosemide-sensitive system. The ouabain-sensitive Na+/K(+)-ATPase is less affected. The transport which is resistant to both inhibitors is not altered by Ha-ras. Overexpression of the Ha-ras proto-oncogene causes only a marginal increase in total 86Rb+ uptake. The stimulation of the furosemide-sensitive influx by Ha-ras is paralleled by an increase in mean cell volume which can be inhibited by furosemide. A rapid stimulation of the furosemide-sensitive Rb+ influx is also observed after addition of bombesin to growth-arrested cells. Furosemide inhibits the mitogenic response after expression of Ha-ras or addition of bombesin. Both the Ha-ras and the bombesin-induced stimulation of the furosemide-sensitive Rb+ transport can be blocked by protein kinase C depletion or the protein kinase C inhibitor staurosporine. In contrast to bombesin-induced phosphatidylinositol-4,5-bisphosphate hydrolysis which is down-modulated by Ha-ras, the stimulation of the furosemide-sensitive Rb+ influx by bombesin is elevated in Ha-ras-expressing cells. This is in accordance with the increased mitogenic activity of bombesin in Ha-ras-expressing cells.     

Studies on bombesin-induced grooming in rats

The gross behavior induced by centrally administered bombesin in rats was compared to that elicited by ACTH-(1–24) and the somatostatin analog, des AA^{1,2,4,5,12,13}[D-Trp⁸]-somatostatin (ODT8-SS). Bombesin (0.001–1 μg, ICV) caused dose-related excessive scratching which was qualitatively different from that associated with the other two groom-inducing agents. Bombesin-induced grooming was not markedly affected by behaviorally nondepressant doses of haloperidol, morphine, naloxone or neurotensin. Bombesin was active in genetically hypotrichotic (essentially furless) rats; and, again in such animals, even after numbing the area caudal to the shoulders with lidocaine. Tolerance and cross-tolerance studies with bombesin and ODT8-SS indicated that they produce scratching through different mechanisms. Bombesin caused scratching when injected directly into the periaqueductal gray, but not when administered intravenously. Neither hypophysectomy nor adrenalectomy markedly affected bombesin-induced grooming. This behavior appears to be initiated in the central nervous system and is produced independently of the pituitary-adrenal axis.     

Suppression of gastric acid secretion by intracisternal bombesin does not require the ventromedial hypothalamus

Intracisternal administration of the tetradecapeptide peptide bombesin suppresses gastric acid release. Other studies have shown that the ventromedial hypothalamus (VMH) may have an inhibitory role in gastric regulation. To determine if the inhibition of gastric acid secretion by intracisternally administered bombesin is mediated by the ventromedial hypothalamus, bombesin was injected intracisternally in rats with ventromedial hypothalamic lesions. Neither anterior nor posterior VMH lesions altered the effects of bombesin on gastric acid, concentration, volume, total output, or on serum gastrin. The bombesin-induced rise in gastric pH was very mildly attenuated by both lesions. The previous finding of enhanced gastric acid secretion after anterior VMH lesions was confirmed. The results suggest that the VMH is not crucial in the bombesin-induced inhibition of acid secretion.     

The effects of neurotensin, naloxone and haloperidol on elements of excessive grooming behavior induced by bombesin

The influence of naloxone, haloperidol and neurotensin was investigated on bombesin-induced excessive grooming in rats. All three drugs reduced the amount of bombesin-induced grooming. Haloperidol induced a general reduction in excessive grooming as induced by bombesin, without changing the composition of grooming behavior, whereas naloxone and neurotensin suppressed bombesin-induced grooming and caused a shift in the distribution of grooming elements. The main suppressive effect of these latter drugs appeared to be on the element scratching. From these data it is suggested that bombesin-induced scratching is mainly displayed by activation of opiate receptor systems, whereas the other elements of bombesin-induced excessive grooming are mainly regulated by dopaminergic systems.     

The cardiovascular effects of a chimeric opioid peptide based on morphiceptin and PFRTic-NH2

MCRT (YPFPFRTic-NH₂) is a chimeric opioid peptide based on morphiceptin and PFRTic-NH₂. In order to assess the cardiovascular effect of MCRT, it was administered by intravenous (i.v.) injection targeting at the peripheral nervous system and by intracerebroventricular (i.c.v.) injection targeting at the central nervous system. Naloxone and l-NAME were injected before MCRT to investigate possible interactions with MCRT. Results show that administration of MCRT by i.v. or i.c.v. injection could induce bradycardia and decrease in mean arterial pressure (MAP) at a greater degree than that with morphiceptin and PFRTic-NH₂. When MCRT and NPFF were coinjected, we observed a dose-dependent weakening of these cardiovascular effects by MCRT. Because naloxone completely abolished the cardiovascular effects of MCRT, we conclude that opioid receptors are involved in regulating the MAP of MCRT regardless of modes of injection. The effect of MCRT on heart rate is completely dependent on opioid receptors when MCRT was administered by i.c.v. instead of i.v. The central nitric oxide (NO) pathway is involved in regulating blood pressure by MCRT under both modes of injection, but the peripheral NO pathway had no effect on lowering blood pressure mediated by MCRT when it was administered by i.c.v. Based on the results from different modes of injection, the regulation of heart rate by MCRT mainly involves in the central NO pathway. Lastly, we observed that the cardiovascular effects of MCRT such as bradycardia and decrease of blood pressure, were stronger than that of its parent peptides. Opioid receptors and the NO pathway are involved in the cardiovascular regulation by MCRT, and their degree of involvement differs between intravenous and intracerebroventricular injection.     

Activation of extracellular signal-regulated kinase mediates bombesin-induced mitogenic responses in prostate cancer cells

Bombesin and its mammalian homologue gastrin-releasing peptide have been shown to be highly expressed and secreted by neuroendocrine cells in prostate cancer, and are thought to be related to the carcinogenesis and progression of this disease. We found, in this study, bombesin specifically induced mitogen-activated protein (MAP) kinase activation as shown by increased extracellular regulated kinase (ERK) phosphorylation and epidermal growth factor (EGF) receptor transactivation in prostate cancer cells, which express functional gastrin-releasing peptide receptor. The transactivation of EGF receptor was required for bombesin-induced ERK phosphorylation. Furthermore, non-receptor tyrosine kinase Src and cellular Ca2+ were shown to be involved in bombesin-induced EGF receptor transactivation and ERK phosphorylation. Inhibition of either EGF receptor transactivation or ERK activation blocked bombesin-induced DNA synthesis in these cells. Taken together, these data suggest bombesin may act as a mitogen in prostate cancer by activating MAP kinase pathway via EGFR transactivation.     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Effects of bombesin on behavior

This report describes the influence of bombesin on the gross behavior of goldfish, frogs, mice, rats, guinea pigs, rabbits, chicks, pigeons and monkeys. Goldfish, frogs, chicks and pigeons were overtly unaffected by bombesin given centrally and/or peripherally. Mice, rats, guinea pigs, rabbits and monkeys responded quickly to intracerebroventricular (i.c.v.) and/or intrathecal (i.th.) administration of bombesin by displaying a range of behaviors suggestive of altered skin sensation. In mice, bombesin was essentially equipotent as a scratch inducer by i.c.v. and i.th. routes (A50 = 0.010-0.019 microgram) but 6800 times less potent i.p. In rats, bombesin-induced grooming and scratching behaviors were shown to be qualitatively different from those associated with ACTH-(1-24) and thyrotropin releasing hormone. Spantide and [D-Arg1, D-Pro2, D-Trp7,9, Leu11]substance P (both at 0.20, 0.50 and 0.80 microgram i.c.v.), two proposed bombesin receptor antagonists, did not markedly influence bombesin-induced scratching or hypothermia in rats.     

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.     

Central mechanisms of acute ANG II modulation of arterial baroreflex control of renal sympathetic nerve activity

Short-term intravenous infusion of angiotensin II (ANG II) into conscious rabbits reduces the range of renal sympathetic nerve activity (RSNA) by attenuating reflex disinhibition of RSNA. This action of ANG II to attenuate the arterial baroreflex range is exaggerated when ANG II is directed into the vertebral circulation, which suggests a mechanism involving the central nervous system. Because an intact area postrema (AP) is required for ANG II to attenuate arterial baroreflex-mediated bradycardia and is also required for maintenance of ANG II-dependent hypertension, we hypothesized that attenuation of maximum RSNA during infusion of ANG II involves the AP. In conscious AP-lesioned (APX) and AP-intact rabbits, we compared the effect of a 5-min intravenous infusion of ANG II (10 and 20 ng x kg(-1) x min(-1)) on the relationship between mean arterial blood pressure (MAP) and RSNA. Intravenous infusion of ANG II into AP-intact rabbits resulted in a dose-related attenuation of maximum RSNA observed at low MAP. In contrast, ANG II had no effect on maximum RSNA in APX rabbits. To further localize the central site of ANG II action, its effect on the arterial baroreflex was assessed after a midcollicular decerebration. Decerebration did not alter arterial baroreflex control of RSNA compared with the control state, but as in APX, ANG II did not attenuate the maximum RSNA observed at low MAP. The results of this study indicate that central actions of peripheral ANG II to attenuate reflex disinhibition of RSNA not only involve the AP, but may also involve a neural interaction rostral to the level of decerebration.     

Pressor doses of vasopressin result in only transient elevations in plasma peptide levels

We recently reported that neuronostatin, a novel neuropeptide, biphasically increased mean arterial pressure, first through the activation of the sympathetic nervous system followed by the release of vasopressin. In those experiments, we found that centrally administered neuronostatin increased plasma vasopressin levels only 2-3 times greater than levels observed in saline-treated controls, and that the increase in mean arterial pressure (approximately 15 mm Hg) could be blocked by pretreatment with a V1-vasopressin antagonist. Here we report the relationship between two to three fold elevations in plasma vasopressin levels and concomitant changes in mean arterial pressure in conscious, unrestrained male rats. We injected increasing doses of vasopressin (5, 20, and 100 ng/kg, intra-arterially) and measured both changes in plasma vasopressin levels and the elevation in mean arterial pressure achieved. At 5-min post injection, plasma levels of vasopressin and mean arterial pressures were similar to those observed following central neuronostatin administration in our earlier study. Thus we conclude that small increases in circulating vasopressin levels can result in significant elevations in mean arterial pressure at least in the conscious rat.     

Corticotropin-releasing factor (CRF): mechanism to elevate mean arterial pressure and heart rate

The efferent mechanisms by which central administration of corticotropin-releasing factor (CRF) elevates mean arterial pressure and heart rate were assessed in unanesthetized, unrestrained rats. CRF increased blood pressure and heart rate by stimulating noradrenergic sympathetic nervous outflow. CRF-induced cardiovascular changes were not dependent on anterior pituitary hormone release, adrenomedullary epinephrine secretion, the renin-angiotensin system or circulating vasopressin.     

Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.

Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.     

Spantide: failure to antagonize bombesin-induced stimulation of gastrin secretion in dogs

Spantide ([d-Arg1, d-Trp7,9, Leu11] substance P) was shown to function not only as a substance P receptor antagonist but also as a bombesin receptor antagonist. This study examined the effects of spantide on intravenous bombesin-induced stimulation of gastrin and acid secretion. Dogs were infused with spantide (1 or 10 nmol kg 1 hr 1) or saline and bombesin (60 pmol kg-1 hr-1), and the gastric acid and plasma gastrin responses were monitored. Spantide did not significantly modify gastrin or gastric acid secretion induced by bombesin. It is concluded that spantide may not be a useful bombesin antagonist for in vivo studies.     

Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.