Comparison of adrenergic and cholinergic receptor-mediated stimulation of gastrin release from rat antral fragments

Rat antral mucosal fragments were maintained in short-term culture to examine the relative potencies and receptor specificity of the cholinergic agonist, carbachol, and adrenergic agents, norepinephrine, isoproterenol, clonidine and phenylephrine in stimulating gastrin release. Results of these studies indicate that norepinephrine and carbachol evoke pharmacologically and temporally distinctive patterns of antral gastrin release. Dose-response experiments indicate that norepinephrine is approximately 10,000 times more potent on a molar basis than carbachol in stimulating antral gastrin release. Adrenergic (norepinephrine, isoproterenol) stimulation of antral gastrin release was prevented by propranolol, and cholinergic- (carbachol) mediated peptide release was blocked by both atropine and pirenzepine. Phenylephrine and clonidine did not alter basal gastrin release. The pattern of peptide release as a function of time was quite different for each agent: norepinephrine exerted its stimulatory effect acutely during the initial 30 minutes of incubation, while carbachol exhibited a sustained stimulatory action throughout the 2-hour culture period. In conclusion, data from these studies suggests that there are marked differences between norepinephrine and carbachol in their pharmacological potency and time-dependent activation of the G cell.     

Beta-adrenergic stimulation of gastrin release mediated by gastrin-releasing peptide in rat antral mucosa

The present studies were directed to examine the effects of beta-adrenergic and cholinergic stimulation on gastrin release and to assess the potential role of gastrin-releasing peptide in exerting these effects, utilizing incubated rat antral mucosa. Rat antral mucosa was incubated at 37 degrees C in Krebs-Henseleit bicarbonate buffer, pH 7.4, continuously gassed with 95% O2-5% CO2. After 1 h media were sampled for radioimmunoassay measurement of gastrin content. Inclusion of carbachol (2.5 X 10(-6) M) in culture medium increased medium gastrin concentration by 106 +/- 28% (P less than 0.01); addition of specific antibodies to gastrin-releasing peptide to the culture medium did not affect carbachol-stimulated gastrin release. Inclusion of isoproterenol (10(-9) M) in culture medium did not affect somatostatin release into the medium, but increased medium gastrin by 234 +/- 24% (P less than 0.001). However, in contrast to carbachol, addition of antibodies to gastrin-releasing peptide to culture medium decreased isoproterenol-stimulated gastrin release by 67 +/- 9% (P less than 0.001). Results of these studies indicate that, under the conditions of these experiments, beta-adrenergic, but not muscarinic, stimulation of gastrin release may be mediated, at least in part, through gastrin-releasing peptide.     

Gastrin secretion by ovine antral mucosa in vitro

The effect on gastrin and somatostatin release in sheep of stimulatory and inhibitory peptides and pharmacological agents was investigated using an in vitro preparation of ovine antral mucosa. Carbachol stimulated gastrin release in a dose-dependent manner but had no effect on somatostatin release. As atropine blocked the effect of carbachol, cholinergic agonists appear to stimulate gastrin secretion directly through muscarinic receptors on the G-cell and not by inhibition of somatostatin secretion. Both vasoactive-intestinal peptide (VIP) and gastric-inhibitory peptide (GIP) increased somatostatin release but did not inhibit basal gastrin secretion, although VIP was effective in reducing the gastrin response to Gastrin-releasing peptide (GRP). Porcine and human GRP were stimulatory to gastrin secretion in high doses but bombesin was without effect. The relative insensitivity to GRP (not of ovine origin) previously reported from intact sheep may be caused either by a high basal release of somatostatin or by the ovine GRP receptor or peptide differing from those of other mammalian species.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Gastrin''s trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

In previous studies we have reported that gastrin exerts a trophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-induced stimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min following exposure to gastrin (10⁻⁸ M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulated activity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observations suggest that protein kinase C mediates the stimulatory effect of gastrin on colonic epithelial cells, possibly through a receptor mechanism.     

Autonomic nervous control of gastric somatostatin secretion from the perfused rat stomach

The effect of parasympathetic and sympathetic nerve stimulation on the secretion of gastric somatostatin and gastrin has been studied in an isolated perfused rat stomach preparation. Stimulation of the vagus nerve inhibited somatostatin secretion and increased gastrin release. Splanchnic nerve stimulation increased somatostatin release during simultaneous atropine perfusion, but not in its absence, whereas gastrin secretion was unchanged. The secretory activity of the gastric D-cell was therefore reciprocally influenced by the sympathetic and parasympathetic nerves but sympathetic stimulation was only effective during muscarinic blockade.     

Effect of cyclic nucleotides on bombesin-evoked gastrin release from isolated perfused rat stomach

We and others have recently reported an involvement of calcium (Ca2+)-mediated intracellular pathways in the release of antral gastrin in response to bombesin (BBS), while cyclic adenosine 3'5'-monophosphate (cAMP) potentiated the gastrin response to BBS. In this study we examined the effect of cyclic nucleotides on BBS-induced gastrin release from isolated perfused rat stomachs. Dibutyryl cyclic AMP (dbcAMP, 1 mM), and Rolipram (a phosphodiesterase inhibitor, 0.5 microM), stimulated basal gastrin secretion and potentiated BBS-induced gastrin release. The stimulation of gastrin release by BBS was not altered by Wiptide (a cAMP dependent protein kinase inhibitor, 1.0 microM), but was surprisingly inhibited by dbcGMP (1 mM). The cAMP content in antral mucosa or in the perfusates was not changed after infusion of BBS. These findings coupled with previous results suggest that BBS-provoked gastrin release is principally coupled to a Ca2+-mediated intracellular pathway, and that an activation of the adenylate cyclase mediated pathway is not involved. Intracellular cGMP, however, may participate in the negative regulation of gastrin release induced by BBS.     

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.     

Effect of thyroid status on alpha- and beta-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased beta-catecholamine responsiveness in hyperthyroid animals is due to a decrease in alpha-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the alpha 2-adrenergic inhibition of adenylate cyclase or the alpha 1-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamsters. No effect of hyperthyroidism was found on the Kd for binding of [3H]dihydroergocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to beta-catecholamines was enhanced in adipocytes from hyperthyroid hamsters, as was lipolysis. However, in adipocytes from hyperthyroid hamsters the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on alpha-catecholamine action by enhances the activation of lipolysis by beta-catecholamine agonists.     

The effect of muscarinic and beta-adrenergic blockade on cysteamine-induced gastrin secretion by the isolated perfused rat stomach

Cysteamine-induced duodenal ulceration in rats is accompanied by increased circulating gastrin. Although cysteamine appears to exert a direct action on the gastrin cell some groups have provided evidence for an involvement of the autonomic nervous system. The current experiments were performed to determine whether beta-adrenergic or cholinergic (muscarinic) pathways are involved in the acute effect of cysteamine on gastrin secretion in the isolated perfused rat stomach. Cysteamine (1 mM) increased gastrin (IRG) secretion to a maximum ranging between 100% and 192% above basal. A cysteamine concentration of 5mM resulted in peak levels ranging between 150% and 1050% above basal. Addition of atropine or propranalol did not influence the responses obtained. The present results, therefore, do not support a role for either cholinergic or beta-adrenergic pathways in cysteamine-induced gastrin release at the level of the stomach and suggest that in vivo such autonomic effects are mediated extrinsically.     

Thyroid hormone stimulation of lipolysis and cyclic adenosine 3', 5'-monophosphate accumulation in human adipose tissue

The role of thyroid hormones on lipolysis in human subcutaneous adipose tissue was investigated. Incubation of subcutaneous fat pads with thyroxine (0.1--10 000 nM) augmented the subsequent isoproterenol stimulation of lipolysis, measured by glycerol release. The basal lipolysis could not by stimulated by thyroxine. The theophylline- and dibutyryl-cyclic AMP stimulated lipolysis also could not be increased by thyroxine at these concentrations. In separate studies, the effect of thyroxine (0.01 pM--1 microM) and triiodothyronine (0.01 pM--1 microM) on cyclic AMP accumulation was examined. No effect of thyroid hormones on cyclic AMP accumulation was seen in non-isoproterenol stimulated tissue. Fat pads stimulated by isoproterenol and then treated with thyroid hormones showed marked increases in accumulation of cyclic AMP as compared to control tissue in the presence of isoproterenol alone.     

The effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6)

Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly stimulated (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin.     

Adrenergic binding sites and enzyme activities in the heart of hyperthyroid rats.

In present study interactions of some adrenergic drugs with the binding of 3H-norepinephrine (NE) and response of some enzymatic systems in the heart of rats with pharmacological hyperthyroidism have been investigated. Binding of NE to cardiac particles was inhibited by isoproterenol, propranolol and in lower concentrations by another beta-blocking drug trimepranol both in control and hyperthyroid hearts in the same degree. However, after addition of nonradioactive norepinephrine (10(-3) M) the degree of displacement was lower in hyperthyroid than in euthyroid group. Activity of adenylate cyclase was lower in hyperthyroid cardiac particles. This difference remained preserved after stimulation by norepinephrine or NaF. The activities of hormone-sensitive lipase and lipoprotein lipase were increased in preparation of hyperthyroid hearts. The phosphorylase "a" activity was also increased in hyperthyroid cardiac particles. There was no change in cardiac adrenergic binding sites properties in hyperthyroidism with the exception of less displacement of NE by nonlabelled hormone. The results indicate that the increased lipolytic and phosphorylase "a" activities in hyperthyroid hearts are not necessarily linked to elevated activity of adenylate cyclase.     

Somatostatin and gastrin release into the gastric lumen in rats

Somatostatin and gastrin release into the gastric lumen was investigated in anaesthetized, vagally intact rats. The stomach was perfused at a flow rate of 0.5 mL.min-1. During perfusion with 0.1 M HCl or buffers of varying pH the somatostatin ans gastrin concentrations in the perfusate were less than 10 pg.mL -1 and approximately 30 pg.mL-1, respectively. Peptone caused a gastrin concentrations in the perfusate were less than 10 pg.mL-1 and approximately 30 pg.mL-1, respectively. Peptone caused a slight pH-independent increase in somatostatin release; gastrin release was unchanged despite an increase in serum gastrin from a basal of 15 +/- 4 to 155 +/- 34 pg.mL-1 during peptone stimulation. intravenous infusion of carbachol (1 microgram.kg-1.min-1) strongly stimulated luminal somatostatin and gastrin release (from 5 +/- 1 to 192 +/- 52 pg.mL-1 and from 27 +/- 5 to 198 +/- 41 pg.mL-1, respectively) during perfusion with 0.1 M HCl. Phosphate buffer perfusion at pH 7.5 abolished the cholinergic-mediated somatostatin release but the gastrin response was unaffected. It is suggested that changes of luminal hormone concentrations in the rat stomach do not reflect the secretory activity of the endocrine cells in the gastric mucosa.     

Progastrin is directed to the regulated secretory pathway by synergistically acting basic and acidic motifs

Bioactivation of prohormones occurs in the granules of the regulated secretory pathway of endocrine cells, which release hormones in response to external stimulation. How secretory granules are formed and how the cargo is selected is still unclear, but it has been shown for several prohormones and processing enzymes that domains within the prohormone structure can act as "sorting signals" for this pathway. The domains mediate interactions with other proteins or with the membrane or facilitate aggregation of the (pro)peptides. We have now searched for domains in progastrin that are active in sorting the prohormone into secretory granules. Truncation studies showed that the N-terminal 30 residues of progastrin are dispensable, whereas the last 49 residues are sufficient for correct biosynthesis of bioactive gastrin. Thus, further N-terminal truncation abolished gastrin expression. C-terminal truncation of 8 residues resulted in an increase in basal secretion as did point mutations in the dibasic processing sites of progastrin. These mutants, however, still responded to secretagogues, suggesting a residual sorting capacity to the regulated pathway. Amino acid substitutions in an acidic, polyglutamate motif within gastrin-17, the main bioactive, cellular gastrin form, did not alter secretion per se, but when these residues were substituted in C-terminally truncated mutants, double mutants increased in basal secretion and did not respond to secretagogue stimulation. This implies that the mutants are constitutively secreted. Our data suggest that the dibasic processing sites constitute the most important sorting domain of progastrin, and these sites act in synergy with the acidic domain.     

The effect of total parenteral nutrition (TPN) on gastrin release in the rat

The effect of short-term (7 days) total parenteral nutrition (TPN) on gastrin release was studied in vivo and in the isolated vascularly perfused rat stomach. The daily plasma gastrin concentration of parenterally fed rats was significantly lower than in ad lib fed control animals (53 +/- 17 pg/ml vs 159 +/- 32 pg/ml, P less than 0.05) as early as day 2 and a similar pattern was observed on days 4 and 6. The fasting plasma gastrin concentration of control animals was 2-fold greater than of the parenterally fed group (P less than 0.05). Following oral peptone, the gastrin response of TPN and control animals doubled although peak gastrin levels were greatly reduced in TPN rats. Basal gastrin release from the perfused stomachs of control rats was 2-fold greater than from TPN rats (P less than 0.05). Electrical stimulation of the vagal trunks resulted in a significantly greater elevation in gastrin secretion from control stomachs compared to TPN animals (4-fold vs. 2.4-fold increase, P less than 0.05). Quantification of the antral G-cell population revealed a significant reduction in the number of G-cell of TPN rats compared to controls (97 +/- 8 cells/mm vs 76 +/- 6 cells/mm, P less than 0.05). These results indicate that luminal nutrient stimulation is necessary for the maintenance of normal G-cell secretory activity in vivo and from the in vitro stomach. G-cell hypoplasia appears to be partially responsible for reduced gastrin output to basal and stimulated conditions after TPN.     

Gastrin release in obese Zucker rats

In this study, gastrin release in the obese Zucker rat was investigated by in vivo and in vitro experiments. Obese rats exhibited normal plasma gastrin levels at 3 weeks (preobese), were moderately hypergastrinemic at 3 months and severely hypergastrinemic at 5 months, compared to lean littermates. Following oral peptone, plasma gastrin levels doubled in both lean and obese rats. Basal and vagally stimulated gastrin release from perfused stomachs was greater in obese compared to lean rats and atropine had no effect on basal gastrin release in either group. Basal somatostatin release from the perfused stomach was found not to differ in both groups of animals. Morphological studies revealed an increase in the number of gastrin-containing G-cells in adult obese rats compared to lean littermates, but not in 3-week-old pups compared to lean littermates, indicating a strong correlation between cell number and plasma gastrin levels. These data indicate that the obese Zucker rat exhibits fasting hypergastrinemia in vivo, a condition which appears after weaning and increases in severity with age. Gastrin hypersecretion persists from the vascularly perfused stomach preparation. The basal hypergastrinemia of the obese Zucker rat is independent of a hyperactive postganglionic cholinergic drive but is associated with and probably causally related to an increase in antral G-cell numbers.     

Modulation of lipolysis by endotoxin and indomethacin in fat cells of germfree and conventional dogs.

Germfree fat cells released significantly less FFA and glycerol under basal conditions (i.e. in the absence of hormonal stimulation) than conventional cells. The lipolytic response to norepinephrine stimulation (0.2 μg/ml) was not different in the two cell populations.$\text{E. coli}$ endotoxin increased basal lipolysis and norepinephrine stimulated (0.2 μg/ml) FFA release in adipocytes from conventional dogs, while having no consistent influence on lipolysis of adipocytes from germfree dogs. The endotoxin effect was not dose dependent (0.2–2.0 μg/0.5 ml cell suspension).Indomethacin (5.0 μg/ml) significantly increased basal FFA and glycerol release from cells of germfree origin, and FFA efflux from cells of conventional dogs. Endotoxin obviated the influence of indomethacin on basal lipolysis of germfree cells.Endotoxin by itself did not alter cAMP concentrations in adipocytes from germfree dogs. The combination of indomethacin and endotoxin, however, significantly increased intracellular cyclic nucleotide concentrations.Compared to conventional fat cells, germfree fat cells are characterized by significantly reduced basal lipolysis, lack of a consistent lipolytic response to endotoxin stimulation and dissociation of the lipolytic response and cAMP levels by the combined influence of endotoxin and indomethacin.     

Functional and anatomical relationships between antral gastrin cells and gastrin-releasing peptide neurons

The present studies were directed to examine the effect of gastrin-releasing peptide (GRP) on beta-adrenergic stimulated gastrin release by cultured rat antral mucosa and to assess the anatomical relationship between gastrin cells and GRP nerves in rat and human antrum. Peptide-containing cells were identified by application of an avidin-biotin-peroxidase immunocytochemical double staining method utilizing antibodies to GRP and gastrin prepared in rabbits. Rat antral mucosa was cultured for 60 min and gastrin released into the culture medium was measured by radioimmunoassay. Inclusion of antibodies to GRP in culture medium did not affect carbachol-stimulated gastrin release, whereas isoproterenol-stimulated gastrin release into the medium was inhibited significantly by addition of GRP antiserum to the culture medium. GRP-containing neurons and axonal fibers were stained immunocytochemically with diaminobenzidine (reddish-brown specific staining) and were located in the lamina propria adjacent to and surrounding the main lobules of antral glands. After double staining utilizing 4-Cl-1-Naphthol as substrate, blue stained gastrin-containing cells were identified in the middle and deeper regions of antral glands in close proximity to GRP neuronal elements. These studies suggest that beta-adrenergic, but not cholinergic, stimulation of gastrin release is mediated, at least in part, through GRP. They also demonstrate intimate anatomical, as well as functional, relationships between gastrin cells and GRP-containing neurons.