I am confused about my teacher's notes. "Acid secretion is stimulated by ACh, gastrin and histamine. Histamine stimulates adenylate cyclase which increases cAMP production."
When I looked this up apparently gastrin decreases AC (adenylate cyclase) production. If histamine and gastrin both stimulate acid secretion why does one increase and the other decrease AC?
Also in her notes the acid secretion inhibitors are acid, protein, fat as they cause the secretion of CCK and secretin and these stimulate AC. But I thought AC stimulated acid secretion not inhibited it?
Wine stimulates gastric acid secretion in isolated rabbit gastric glands via two different pathways
Alcoholic beverages such as beer and wine are well known to potently stimulate gastric acid secretion, most probably through an increase in circulating gastrin level. The present study examined whether or not wine stimulates gastric acid secretion by a direct effect on parietal cells, enterochromaffin-like (ECL) cells or both.
Gastric mucosa was isolated from female Japanese white rabbits and gland specimens were prepared by the collagenase digestion method. Acid secretion was assessed by gland accumulation of [ 14 C] aminopyrine. The effects of red wine, ethanol, non-alcoholic wine and drugs were determined by incubating gastric glands with aminopyrine. Radioactivity in solubilized glands was determined by a liquid scintillation counting.
Neither wine nor ethanol (diluted 1 : 10 2 to 1 : 10 4 ) had any effect on gastric acid secretion, whereas non-alcoholic wine stimulated acid secretion in a dose-dependent manner. All substances, however, significantly stimulated gastric acid secretion in IBMX (phosphodiesterase inhibitor)-pretreated glands. S-0509 (a CCK-2 receptor antagonist) and atropine had no effect on acid secretion stimulated by wine, ethanol or non-alcoholic wine in IBMX-pretreated glands. Famotidine and omeprazole significantly inhibited the acid secretion resulting from all of the above stimulants. BAPTA (an intracellular Ca 2+ chelator) inhibited acid secretion stimulated with wine or ethanol in a dose-dependent manner, but did not inhibit secretion stimulated by non-alcoholic wine.
Wine was found to stimulate gastric acid secretion in gastric glands via two pathways, by an ethanol-induced increase in the concentration of intracellular Ca 2+ in parietal cells, and by histamine release from ECL cells potentially induced by constituents present in wine.
Parietal cells are responsible for gastric acid secretion, which aids in the digestion of food, absorption of minerals, and control of harmful bacteria. However, a fine balance of activators and inhibitors of parietal cell-mediated acid secretion is required to ensure proper digestion of food, while preventing damage to the gastric and duodenal mucosa. As a result, parietal cell secretion is highly regulated through numerous mechanisms including the vagus nerve, gastrin, histamine, ghrelin, somatostatin, glucagon-like peptide 1, and other agonists and antagonists. The tight regulation of parietal cells ensures the proper secretion of HCl. The H + -K + -ATPase enzyme expressed in parietal cells regulates the exchange of cytoplasmic H + for extracellular K + . The H + secreted into the gastric lumen by the H + -K + -ATPase combines with luminal Cl − to form gastric acid, HCl. Inhibition of the H + -K + -ATPase is the most efficacious method of preventing harmful gastric acid secretion. Proton pump inhibitors and potassium competitive acid blockers are widely used therapeutically to inhibit acid secretion. Stimulated delivery of the H + -K + -ATPase to the parietal cell apical surface requires the fusion of intracellular tubulovesicles with the overlying secretory canaliculus, a process that represents the most prominent example of apical membrane recycling. In addition to their unique ability to secrete gastric acid, parietal cells also play an important role in gastric mucosal homeostasis through the secretion of multiple growth factor molecules. The gastric parietal cell therefore plays multiple roles in gastric secretion and protection as well as coordination of physiological repair.
This review summarizes the complex literature related to the physiology and cell biology of gastric parietal cell acid secretion and the impact of directed pharmacology in the therapeutic manipulation of acid secretion. In addition, the article addresses the role of gastric parietal cells as sources of growth factors and regulators of gastric mucosal homeostasis.
Materials and methods
Histamine, carbachol (carbamylcholine chloride), recombinant human TNF-α, recombinant human IL-1β, gastrin-17, Earle’s balanced salt solution (EBSS), A23187, bovine serum albumin (BSA), pertussis toxin, collagenase type I, collagenase type H, dithiothreitol, penicillin G, crystalline bovine insulin, hydrocortisone, streptomycin, gentamicin, dinitrophenol, and EDTA were purchased from Sigma, Poole, UK. Forskolin, herbimycin A, N 6 ,O 2 -dibutyryladenosine 3, 5-cyclic monophosphate (dibutyryl cyclic AMP: dbcAMP) were obtained from LC laboratories, Nottingham, UK Triton X-100 and N-2-hydroxyethylpiperazine-N′-ethansulphonic acid (HEPES) were from BDH-Merck (Poole, UK) 14 C-dimethylamine-aminopyrine (103 mCi/mmol) was obtained from Amersham International, Amersham, UK and 125 I-CCK-8 (2200 Ci/mmol) and 3 H-histamine (50 Ci/mmol) from Dupont-NEN, Stevenage, UK. Ham’s F12/Dulbecco’s modified Eagle’s culture media (F12/DMEM) (50:50 vol/vol), glutamine, Hank’s balanced salt solution (HBSS), basal medium Eagle’s (BME), and fetal calf serum were obtained from Gibco, Paisley, UK basement membrane Matrigel was from Universal Biologicals, London, UK and ranitidine was from Glaxo, Uxbridge, UK.
PARIETAL CELL PREPARATION
Rabbit parietal cells were isolated and enriched by a modification of previously described methods.17-19 New Zealand white rabbits (body weight 2–2.5 kg) were killed by overdose of sodium pentobarbitone and the stomach immediately excised. Gastric fundic mucosa was digested with sequential exposure to collagenase (type I 0.175 g/l with type H 0.175 g/l) and EDTA. Parietal cells were enriched from the crude suspension with a Beckman JE 5.0 elutriator rotor using the standard elutriation chamber.17 For selected experiments further purification of parietal cells was performed using density gradient centrifugation with 50% percoll as previously described.20
Cells from the parietal cell enriched fractions were collected by brief centrifugation and pooled and resuspended in complete culture medium (Ham’s F12/DMEM 50/50 nutrient mix, containing 10% heat inactivated fetal calf serum, 10 mM HEPES pH 7.4, 100 mg/l gentamicin, 100 mg/l streptomycin, 100 mg/l penicillin, 2 mM glutamine, 8 μg/ml hydrocortisone, and 1 μg/ml insulin) and then cultured with a modification of previously described methods.19 Parietal cells were then plated on to Matrigel coated 12 and 24 well tissue culture plates (Corning) at 0.5–1.0 million cells/well. The coated plates had been prepared by diluting Matrigel 1:7 with sterile water and then uniformly coating the wells. After setting with overnight incubation at 37°C, the remaining water was aspirated and the wells were allowed to dry in a culture hood for 60 minutes. The plates were then equilibrated with 1 ml culture medium before plating the cells. The parietal cell enriched fraction was then cultured at 37°C in an atmosphere of 5% CO2/95% air for 40 hours.
MEASUREMENT OF ACID SECRETION
Intracellular accumulation of 14 C-aminopyrine was used as a measure of functional acid secretory activity.18-21Cultured cells in 24 well plates were washed once with 2 ml EBSS containing 0.1 % BSA, 10 mM HEPES pH 7.4, 2 mM glutamine, and 0.22% NaHCO3 to remove dead and non-adherent cells and then 1 ml of the above medium was added and 0.1 μCi aminopyrine and the various stimulant substances were added simultaneously to each well. Cells were then incubated for 30 minutes at 37°C in an atmosphere of 5% CO2/95% air. Incubations were terminated by removing the medium from each well using a vacuum pump and then washing twice with 1 ml EBSS solution. Cells were then lysed with 1 ml 1% Triton X-100. Aliquots of cell lysates and incubation media were counted in Optiphase Safe, Wallac, Milton Keynes, UK, using a Beckman LS 1801 liquid scintillation counter with DPM correction. Dinitrophenol (0.1 mM) was added to separate wells to assess non-specific incorporation and values were subtracted from test values.13 For most of the studies with recombinant cytokines, the TNF-α and IL-1β were added to the wells 15 minutes before the initial washing step and before adding the aminopyrine and stimulants. Pilot studies showed that this method gave results corresponding to those obtained if the cells were washed first and then cytokines added and preincubated for 15 minutes before being stimulated. For time course experiments the cytokines were added to the culture medium at intervals up to 18 hours before aminopyrine accumulation was measured simultaneously in all test wells. When appropriate, pertussis toxin (200 ng/ml) was added two hours before and herbimycin was added one hour before the cytokines.
RECEPTOR BINDING STUDIES
Binding of 3 H-histamine to H2 receptors and 125 I-CCK-8 to gastrin/CCKB receptors on parietal cells was assessed as previously described (labelled CCK-8 has been shown previously to bind with equal efficacy as 125 I-[Leu 15 ]gastrin to parietal cell CCKB receptors and was used as tracer because of its ready availability).22 , 23 Briefly, cells on Matrigel coated 12 well plates were washed three times with HBSS containing 0.1% BSA to remove non-adherent cells and incubated at 37°C with 10 fmol 125 I-Bolton-Hunter CCK-8 for 60 minutes or 1 pmol 3 H-histamine for 30 minutes in the presence or absence of unlabelled gastrin or the H2-receptor antagonist ranitidine as appropriate. Binding reactions were terminated by aspirating the medium containing the unbound label and then the cells were washed twice with ice chilled HBSS. The adherent cells were solubilised by incubating in 1% Triton X-100 and the bound and unbound radioactivity was counted in a gamma counter (Wallac 1260, Wallac, Milton Keynes, UK) or scintillation counter as necessary. Binding of 125 I-CCK in the presence of 10 -6 M gastrin and 3 H-histamine binding in the presence of 10 -4 M ranitidine was regarded as non-specific binding and these values were subtracted from the total binding to obtain the values for specific binding.
During each set of experiments, each condition was assessed in duplicate wells and compared with basal (unstimulated) and the appropriate stimulated aminopyrine accumulation from wells on the same 24 well plate. The mean of the data from one animal preparation was regarded as n=1. For time course experiments the effects of cytokines were compared with parietal cells on the same 24 well plate treated identically but without addition of cytokine. Data are presented as the mean (SEM) of 3–12 different animal preparations. Graphical data are presented as aminopyrine accumulation compared with basal values to enable comparison of the relative stimulation seen with the different agonists. Student’s paired t test was used to compare the effect of cytokines with the appropriate controls. A p value <0.05 was regarded as significant.
Properties of isolated gastric enterochromaffin-like cells.
The gastric enterochromaffin-like cell (ECL) has been studied in gastric fundic glands by confocal microscopy and as a purified cell preparation by video imaging of calcium signaling and measurements of histamine release. Regulation of gastric acid secretion is largely due to alterations of histamine activation of the H2 receptor on the parietal cell and can be divided into central neural regulation, with direct actions of neuronally released mediators and into peripheral regulation by substances released from other endocrine cells. Gastric neuronal stimulation of acid secretion by alteration of ECL cell function is probably mediated by pituitary adenylate cyclase activating peptide (PACAP) receptors on the ECL cell, which activate calcium signaling and histamine release. Peripheral stimulation of acid secretion via the ECL cell is largely mediated by gastrin stimulation of calcium signaling and histamine release. Gastric neuronal inhibition of ECL cell function is probably mediated by galanin inhibition of calcium signaling, and histamine release and peripheral inhibition of ECL cell function is mainly due to somatostatin release from D cells.
INTRACELLULAR SIGNALING MECHANISMS
As in other tissues, PACAP potently increases cellular content of cAMP in insulin-producing cells, as evident from studies in clonal HIT-T15 cells in which a marked increase in cAMP content is apparent after only a 2-min incubation with PACAP38, and PACAP-induced insulin secretion is inhibited by H89, an inhibitor of protein kinase (PK)-A (67,69,82). In some cell systems, it has been shown that PACAP acts not only through AC but also through the PLC pathway, with an activation of PKC and an increased formation of intracellular inositol phosphates (IPs) (26). However, the insulinotropic effect of PACAP is not diminished by downregulation of PKC by the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (69), which suggests that PKC is not of major relevance for the insulinotropic action of PACAP. Similarly, PACAP only slightly increases IP3 formation in insulin-producing cells, suggesting that this pathway is also of limited relevance (67).
In both islet and clonal HIT-T15 cells, PACAP increases the cytoplasmic concentration of calcium by an effect that is abolished by either removal of extracellular calcium or calcium channel blockade (61,67,69). This suggests that increased cytoplasmic calcium is caused by the opening of membrane-bound calcium channels. Electrophysiological studies in HIT-T15 cells have shown that PACAP also increases cytoplasmic calcium by releasing calcium from intracellular stores, which is an action dependent on a small prior influx of calcium through membrane-bound channels (83). However, stimulated calcium influx by PACAP is mainly of importance for its insulinotropic action because the removal of extracellular calcium markedly reduces PACAP-stimulated insulin secretion (67). Because PACAP-induced formation of cAMP in HIT-T15 cells is not markedly affected by the removal of extracellular calcium at the same time (K.F., B.A., unpublished observations), it is assumed that the formation of cAMP (through activation of PKA) induces calcium uptake, as previously observed (84). This increase in cytoplasmic calcium subsequently transduces the potent insulinotropic response of PACAP.
PACAP has also been shown to open membraneous sodium channels, causing an inward membrane current (83), and studies using fluorophor-labeled clonal cells have displayed an increase in the cytoplasmic sodium concentration after administration of PACAP (85). Furthermore, removal of extracellular sodium impairs the insulinotropic action of PACAP without affecting the formation of cAMP (85), suggesting that the uptake of sodium is involved in PACAP-induced insulin secretion. The increase in cytoplasmic sodium by PACAP has been shown to be abolished by the PKA inhibitor H89, suggesting that the sodium channel is regulated by cAMP and PKA (85). The influx of sodium might contribute to the opening of the voltage-dependent calcium channels through a depolarizing effect, thus increasing cytoplasmic calcium and thereby potentiating insulin secretion (83). However, PACAP has been shown to increase cytoplasmic calcium in the absence of extracellular sodium (85), suggesting that sodium is also of importance for the events of exocytosis distal to calcium influx. However, this needs further investigation. One study has shown that wortmannin, which inhibits phosphatidylinositol 3-kinase (PI 3-K), partially inhibits PACAP-induced insulin secretion in HIT-T15 cells, suggesting that this pathway is also involved in the mediation of the insulinotropic effect of PACAP (69). However, it was simultaneously shown that PACAP-induced insulin secretion is not accompanied by increased PI 3-K activity, implying that a PI 3-K–independent, yet wortmannin-sensitive, signaling pathway is involved in the insulinotropic action of PACAP (69). This pathway may involve distal mechanisms that affect the exocytotic expulsion of granules. Hence, although more studies are required for elucidating the exact β-cell mechanism of PACAP, most evidence suggests that PACAP induces insulin secretion mainly by increasing cellular cAMP and activating PKA in association with the uptake of extracellular cations (Fig. 4).
Does adenylate cyclase stimulate or inhibit acid secretion in the stomach? - Biology
N-alpha-methyl-histamine, which is produced in the gastric mucosa by Helicobacter pylori, is a potent H2 receptor agonist as well as a H3 receptor agonist
It is over 80 years since the stimulatory effect of histamine on gastric acid was reported. 1 The observation that the conventional antihistamines (subsequently shown to be H1 antagonists) failed to block the acid stimulatory action 2 ultimately led to the discovery and availability of H2 antagonists. 3 These were not only effective drugs but tools to dissect acid secretory physiology, and develop our continually evolving paradigm of histamine as the major paracrine stimulant of gastric acid. 4
In the gastric mucosa, histamine is found within enterochromaffin-like (ECL) cells and mast cells, the relative proportion of the two cell types being species and site dependent. Histamine is formed by the decarboxylation of histamine by histidine decarboxylase (HDC). After release histamine is enzymatically deactivated by two pathways. The majority is methylated onto one of the nitrogen atoms in the imidazole ring by imidazole-N-methyltransferase, and a smaller proportion is degraded by oxidative deamination to imidazole acetic acid. A further potential methylation site is on the terminal nitrogen of the side chain, producing N-alpha-methyl-histamine (NAMH). In 1935, soon after it was first chemically synthesised, NAMH was shown to be a potent stimulant of canine acid secretion. 5 NAMH was detected in canine gastric juice following histamine stimulation, and was more than twice as potent as histamine in stimulating acid secretion. 6 The acid stimulatory action was sensitive to H2 blockers. Although a broad specificity mammalian enzyme capable of catalysing side chain as well as ring methylation was subsequently described, 7 there has never been any evidence that NAMH was a physiological product.
It is likely that the gastric effects of NAMH would have been sidelined if it were not for two independent discoveries in the early 1980s. The first was the description of Helicobacter pylori, as we now know it, and the subsequent interest in gastric physiology. The second was the description of a novel high affinity histamine receptor type (H3). 8 Initially described as receptors inhibiting histamine release in rat brain, many studies followed characterising the receptor. It was soon appreciated that H3 receptor agonists could inhibit acid secretion in vivo, 9 inhibit histamine secretion from ECL cells in vitro, 10 and possibly regulate gastrin and somatostatin secretion. 9 NAMH was found to be a high affinity H3 receptor agonist and it came to be used widely as a ligand to investigate the H3 receptor. 9
Courillon-Mallet et al brought these two strands together NAMH and N-alpha-methylating activity were detected by biochemical means in H pylori positive mucosa and cultures of H pylori, but not in H pylori negative mucosa. Binding studies suggested bacterially produced NAMH was occupying mucosal H3 receptors. This occupation seemed to be correlated with suppression of both HDC activity and mucosal somatostatin. 11
NAMH became an attractive putative mediator of the abnormalities of gastric secretion in H pylori infection. NAMH stimulated acid secretion in cultured isolated parietal cells 12 and gastrin secretion from cultured G cells 13 : these effects were blocked by H2 antagonists. No evidence for NAMH acting on H3 receptors inhibiting either parietal cell acid secretion or D cell somatostatin secretion were found. 12, 14
In this issue of Gut, Saitoh et al have examined the interaction of NAMH with the H2 receptor in an attempt to clarify these disparate observations [see 786]. 15 They utilised a Chinese hamster ovary cell line stably transfected with, and expressing, the human H2 receptor gene. Histamine and NAMH displaced tiotidine (a H2 antagonist) from the receptor but the archetypal H3 selective agonist (R)-alpha-methyl-histamine did not. Functional activation of the H2 receptor by both histamine and NAMH was illustrated by dose dependent cAMP generation. This was blocked by the H2 antagonist famotidine but not by the H3 antagonist thioperamide. NAMH demonstrated greater potency in terms of cAMP generation, with greater maximal response and lower EC50, while histamine exhibited more affinity for the receptor, as demonstrated by radioligand displacement. Transfectant studies are a powerful means of characterising receptors and these studies have confirmed the inferences drawn from previous investigations that NAMH is a potent agonist at both H2 and H3 receptors.
Saitoh et al studied coupling to adenylate cyclase, which is believed to be the acid stimulatory pathway in parietal cells, as the marker for receptor activation. It is now known that H2 receptors can also directly couple to the phosphoinositide signalling pathway and activate the protein kinase C, mitogen activated protein kinase, and p70 S6 kinase pathways. 16, 17 Activation of these pathways may be involved in H2 receptor dependent regulation of growth and differentiation. In view of the proliferative effects of H pylori infection and the suggestion that H2 receptor agonism stimulates growth of gastric carcinoma cells, 18 it would have been interesting if Saitoh et al had studied these interactions.
Two notes of caution must be raised when considering the results of this study. Although in retrospect the source of canine gastric NAMH was likely to have been Helicobacter rather than canine metabolism, the potential role of NAMH in human pathophysiology requires further assessment. Only small numbers have been studied and the data are inconsistent. NAMH was detected in the gastric juice of 5/7 H pylori positive and 0/9 H pylori negative subjects using gas chromatography-mass spectrophotometry, 19 but in contrast with Courillion-Mallet and colleagues, 11 it was not detected in either H pylori positive gastric biopsies or cultures of H pylori.
Secondly, the physiological role of histamine receptors in the stomach does not appear to be the simple balance outlined by Saitoh et al. While activation of H2 receptors by histamine and NAMH explains the acid stimulatory actions, data concerning potentially inhibitory actions of the H3 receptor are conflicting. The majority of in vivo studies have confirmed that (R)-alpha-methyl-histamine inhibits acid secretion. The data are most consistent with inhibitory H3 receptors located on ECL cells and cholinergic or intramural neurones, 9, 14 although increased acid secretion secondary to reduced somatostatin secretion has been reported. 20 At present it is not clear whether these differences reflect species or methodological variation. Application of knowledge from the cloning of the H3 receptor gene should clarify this situation. 21
The recent description of a fourth receptor type 22 emphasises the fact that we still have much to learn about the biology of this deceptively simple molecule.
N-alpha-methyl-histamine, which is produced in the gastric mucosa by Helicobacter pylori, is a potent H2 receptor agonist as well as a H3 receptor agonist
REVIEW articleDora Reglodi 1 *, Anita Illes 1,2 , Balazs Opper 1 , Eszter Schafer 3 , Andrea Tamas 1 and Gabriella Horvath 1
- 1 Department of Anatomy, MTA-PTE PACAP Research Team, Centre for Neuroscience, University of Pecs Medical School, Pecs, Hungary
- 2 1st Department of Internal Medicine, University of Pecs Medical School, Pecs, Hungary
- 3 Department of Gastroenterology, Medical Centre, Hungarian Defence Forces, Budapest, Hungary
Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with widespread occurrence throughout the body including the gastrointestinal system. In the small and large intestine, effects of PACAP on cell proliferation, secretion, motility, gut immunology and blood flow, as well as its importance in bowel inflammatory reactions and cancer development have been shown and reviewed earlier. However, no current review is available on the actions of PACAP in the stomach in spite of numerous data published on the gastric presence and actions of the peptide. Therefore, the aim of the present review is to summarize currently available data on the distribution and effects of PACAP in the stomach. We review data on the localization of PACAP and its receptors in the stomach wall of various mammalian and non-mammalian species, we then give an overview on PACAP’s effects on secretion of gastric acid and various hormones. Effects on cell proliferation, differentiation, blood flow and gastric motility are also reviewed. Finally, we outline PACAP’s involvement and changes in various human pathological conditions.
Materials and Methods
Experiments were performed using adult male (10–30 weeks of age) and female (5–30 weeks of age) suncus of an outbred KAT strain established from a wild population in Kathmandu, Nepal , weighing between 50 and 100 g. Animals were housed individually in plastic cages equipped with an empty can for a nest box under controlled conditions (23 ± 2°C, lights on from 8:00 to 20:00) with free access to water and commercial feeding pellets (number 5P Nippon Formula Feed Manufacturing, Yokohama, Japan). The metabolizable energy content of the pellets was 344 kcal/100 g. The pellets consisted of 54.1% protein, 30.1% carbohydrates, and 15.8% fat. All procedures were approved and performed in accordance with the Committee on Animal Research of Saitama University (Saitama, Japan). All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiment.
Gastric acid secretion was stimulated by intravenous (i.v.) bolus infusion of histamine dihydrochloride (Nakarai Chemicals Co., Ltd., Kyoto, Japan), pentagastrin (Sigma Aldrich, USA), carbachol (Tocris Bioscience, Ellisville, MO), suncus motilin (Scrum Inc., Tokyo, Japan), and human acylated ghrelin (Asubio Pharma Co., Ltd., Hyogo, Japan). Famotidine (Wako Pure Chemical Industries Ltd.), YM 022 (Sigma-Aldrich), and atropine sulfate (Mylan Pharma, Osaka, Japan) were used as H2 receptor, CCK-B receptor, and muscarinic cholinergic receptor antagonist, respectively. Motilin, ghrelin, and pentagastrin were dissolved in 0.1% BSA in PBS (phosphate-buffered saline), while histamine, carbachol, and atropine were dissolved in 0.9% saline. Famotidine was dissolved in 0.5 N HCl, and i.v. administration was performed after dilution in 0.1% BSA in PBS. YM 022 was dissolved in dimethylsulfoxide (DMSO) and subsequent dilutions were made in saline containing DMSO. All solutions were prepared immediately before each experiment. The dosage of each agent was administered at a rate of 100 μL per 100 g BW.
Determination of gastric acid output by an intragastric perfusion experimental system
Overnight-fasted animals were anesthetized with an intraperitoneal injection of 15% urethane solution at a dose of 1 ml/100 g body weight, and prepared for the gastric acid output study by using intragastric perfusion system (S1 Fig). After anesthesia, the animals' tracheas were exposed, cannulated, and exteriorized to avoid choking due to catheter insertion through esophagus. Then, a catheter (polyethylene tube) was inserted into the stomach from the mouth and the tip of the catheter was placed about 3 mm from the cardia inside the stomach the tube was fixed to the esophagus with a suture. Then, the abdomen was opened through the linea alba to expose the stomach. The pyloroduodenal junction was then exposed, and another polyethylene tube was introduced into the stomach via an incision in the duodenum, and was secured firmly with a ligature around the pylorus. Then, we exposed the jugular vein to insert a cannula for administration of reagents/drugs. The stomach lumen was washed with saline until the effluent was clear and then perfused with saline solution at 37°C at a rate of 0.25 ml/min using a peristaltic pump (Micro tube pump MP-3EYELA Tokyo Rikakikai Co., Ltd., Tokyo). The stomach was perfused with saline through the liquid discharge tube and gastric output was collected through the perfusion catheter. In order to stabilize the amount of acid secretion, the pH of the collected solution was allowed to stabilize for 60 minutes from the start of the perfusion of saline. Effluents were collected in the tube continuously at 10-min intervals by a DF-2000 fraction collector (Tokyo Rikakikai Co., Ltd., Tokyo, JAPAN). We measured the pH of the gastric output with a pH meter (HORIBA Scientific, LAQUA ELECTRODE, HORIBA Ltd., JAPAN). We determined the acid output by a neutralization titration using 0.01 N NaOH solution. The amount of acid secretion was expressed per 10 minutes as H + μEq. The amount of change in gastric secretion was measured by deducting the area under the curve (AUC) of the gastric acid secretion at 50 min before and after the administration of each drug. The values are expressed as ΔμEq/50 min.
Intravenous (i.v.) bolus infusion of histamine dihydrochloride at the dose of 1 mg/kg BW , human acylated ghrelin (0.1, 1, and 10 μg/kg BW), and suncus motilin (0.1, 1, and 10 μg/kg BW) was done to study their stimulatory effect on acid secretion. A previous study confirmed that seven amino acids with acyl modification on the third Ser residue in the N-terminal region of ghrelin showed full biological activity . In addition, the first 10 amino acids of mature suncus ghrelin sequence are identical among other mammals, including humans , indicating that human ghrelin is enough to exert full biological activity in suncus. Therefore, we used human ghrelin in this experiment. Combined effects of motilin and ghrelin were studied by the co-administration of motilin and ghrelin at doses of 0.1, 1, and 10 μg/kg BW (each). Based on the dose response, motilin and co-administration of motilin and ghrelin at 10 μg/kg BW were selected for all further studies. Famotidine (0.33 mg/kg BW)  was used to evaluate the role of the histamine (H2) receptor on motilin and co-administration of motilin and ghrelin stimulating gastric acid secretion. After confirming that pretreatment of famotidine (0.33 mg/kg BW) completely inhibited histamine-stimulated (1 mg/kg BW) acid secretion, famotidine at the pretreatment dose was administered 30 min before administration of motilin (10 μg/kg BW) and co-administration of motilin (10 μg/kg BW) and ghrelin (10 μg/kg BW). To examine the involvement of gastrin (CCK-B) receptors in motilin-stimulated gastric acid secretion, we also administered YM 022 (0.2 mg/kg BW) [36, 37]. Vehicle or YM 022 (0.2 mg/kg BW) was administered 30 min before each drug injection. Similarly, an mACh receptor antagonist, atropine (30 μg/kg BW) was also administered 30 min before motilin or motilin/ghrelin co-administration to examine the influence of these receptors on gastric acid secretion.
We repeated the recording experiments individually at least three times. All data are indicated as mean ± S.E.M. We used GraphPad Prism 5 software (GraphPad Software Inc., CA, USA) to analyze the data. Statistical analyses were performed using a one-way analysis of variance (ANOVA) followed by Dunnett's Multiple Comparisons Test or Student’s t test. p < 0.05 was considered statistically significant.
BIO 140 - Human Biology I - Textbook
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- Identify the three major classes of hormones on the basis of chemical structure
- Compare and contrast intracellular and cell membrane hormone receptors
- Describe signaling pathways that involve cAMP and IP3
- Identify several factors that influence a target cell&rsquos response
- Discuss the role of feedback loops and humoral, hormonal, and neural stimuli in hormone control
Although a given hormone may travel throughout the body in the bloodstream, it will affect the activity only of its target cells that is, cells with receptors for that particular hormone. Once the hormone binds to the receptor, a chain of events is initiated that leads to the target cell&rsquos response. Hormones play a critical role in the regulation of physiological processes because of the target cell responses they regulate. These responses contribute to human reproduction, growth and development of body tissues, metabolism, fluid, and electrolyte balance, sleep, and many other body functions. The major hormones of the human body and their effects are identified in Table 1.
Table 1: Endocrine Glands and Their Major Hormones
|Endocrine gland||Associated hormones||Chemical class||Effect|
|Pituitary (anterior)||Growth hormone (GH)||Protein||Promotes growth of body tissues|
|Pituitary (anterior)||Prolactin (PRL)||Peptide||Promotes milk production|
|Pituitary (anterior)||Thyroid-stimulating hormone (TSH)||Glycoprotein||Stimulates thyroid hormone release|
|Pituitary (anterior)||Adrenocorticotropic hormone (ACTH)||Peptide||Stimulates hormone release by adrenal cortex|
|Pituitary (anterior)||Follicle-stimulating hormone (FSH)||Glycoprotein||Stimulates gamete production|
|Pituitary (anterior)||Luteinizing hormone (LH)||Glycoprotein||Stimulates androgen production by gonads|
|Pituitary (posterior)||Antidiuretic hormone (ADH)||Peptide||Stimulates water reabsorption by kidneys|
|Pituitary (posterior)||Oxytocin||Peptide||Stimulates uterine contractions during childbirth|
|Thyroid||Thyroxine (T4), triiodothyronine (T3)||Amine||Stimulate basal metabolic rate|
|Thyroid||Calcitonin||Peptide||Reduces blood Ca 2+ levels|
|Parathyroid||Parathyroid hormone (PTH)||Peptide||Increases blood Ca 2+ levels|
|Adrenal (cortex)||Aldosterone||Steroid||Increases blood Na + levels|
|Adrenal (cortex)||Cortisol, corticosterone, cortisone||Steroid||Increase blood glucose levels|
|Adrenal (medulla)||Epinephrine, norepinephrine||Amine||Stimulate fight-or-flight response|
|Pineal||Melatonin||Amine||Regulates sleep cycles|
|Pancreas||Insulin||Protein||Reduces blood glucose levels|
|Pancreas||Glucagon||Protein||Increases blood glucose levels|
|Testes||Testosterone||Steroid||Stimulates development of male secondary sex characteristics and sperm production|
|Ovaries||Estrogens and progesterone||Steroid||Stimulate development of female secondary sex characteristics and prepare the body for childbirth|
Types of Hormones
The hormones of the human body can be divided into two major groups on the basis of their chemical structure. Hormones derived from amino acids include amines, peptides, and proteins. Those derived from lipids include steroids (Figure 1). These chemical groups affect a hormone&rsquos distribution, the type of receptors it binds to, and other aspects of its function.
Hormones derived from the modification of amino acids are referred to as amine hormones. Typically, the original structure of the amino acid is modified such that a &ndashCOOH, or carboxyl, group is removed, whereas the , or amine, group remains.
Amine hormones are synthesized from the amino acids tryptophan or tyrosine. An example of a hormone derived from tryptophan is melatonin, which is secreted by the pineal gland and helps regulate circadian rhythm. Tyrosine derivatives include the metabolism-regulating thyroid hormones, as well as the catecholamines, such as epinephrine, norepinephrine, and dopamine. Epinephrine and norepinephrine are secreted by the adrenal medulla and play a role in the fight-or-flight response, whereas dopamine is secreted by the hypothalamus and inhibits the release of certain anterior pituitary hormones.
Peptide and Protein Hormones
Whereas the amine hormones are derived from a single amino acid, peptide and protein hormones consist of multiple amino acids that link to form an amino acid chain. Peptide hormones consist of short chains of amino acids, whereas protein hormones are longer polypeptides. Both types are synthesized like other body proteins: DNA is transcribed into mRNA, which is translated into an amino acid chain.
Examples of peptide hormones include antidiuretic hormone (ADH), a pituitary hormone important in fluid balance, and atrial-natriuretic peptide, which is produced by the heart and helps to decrease blood pressure. Some examples of protein hormones include growth hormone, which is produced by the pituitary gland, and follicle-stimulating hormone (FSH), which has an attached carbohydrate group and is thus classified as a glycoprotein. FSH helps stimulate the maturation of eggs in the ovaries and sperm in the testes.
The primary hormones derived from lipids are steroids. Steroid hormones are derived from the lipid cholesterol. For example, the reproductive hormones testosterone and the estrogens&mdashwhich are produced by the gonads (testes and ovaries)&mdashare steroid hormones. The adrenal glands produce the steroid hormone aldosterone, which is involved in osmoregulation, and cortisol, which plays a role in metabolism.
Like cholesterol, steroid hormones are not soluble in water (they are hydrophobic). Because blood is water-based, lipid-derived hormones must travel to their target cell bound to a transport protein. This more complex structure extends the half-life of steroid hormones much longer than that of hormones derived from amino acids. A hormone&rsquos half-life is the time required for half the concentration of the hormone to be degraded. For example, the lipid-derived hormone cortisol has a half-life of approximately 60 to 90 minutes. In contrast, the amino acid&ndashderived hormone epinephrine has a half-life of approximately one minute.
Pathways of Hormone Action
The message a hormone sends is received by a hormone receptor , a protein located either inside the cell or within the cell membrane. The receptor will process the message by initiating other signaling events or cellular mechanisms that result in the target cell&rsquos response. Hormone receptors recognize molecules with specific shapes and side groups, and respond only to those hormones that are recognized. The same type of receptor may be located on cells in different body tissues, and trigger somewhat different responses. Thus, the response triggered by a hormone depends not only on the hormone, but also on the target cell.
Once the target cell receives the hormone signal, it can respond in a variety of ways. The response may include the stimulation of protein synthesis, activation or deactivation of enzymes, alteration in the permeability of the cell membrane, altered rates of mitosis and cell growth, and stimulation of the secretion of products. Moreover, a single hormone may be capable of inducing different responses in a given cell.
Pathways Involving Intracellular Hormone Receptors
Intracellular hormone receptors are located inside the cell. Hormones that bind to this type of receptor must be able to cross the cell membrane. Steroid hormones are derived from cholesterol and therefore can readily diffuse through the lipid bilayer of the cell membrane to reach the intracellular receptor (Figure 2). Thyroid hormones, which contain benzene rings studded with iodine, are also lipid-soluble and can enter the cell.
The location of steroid and thyroid hormone binding differs slightly: a steroid hormone may bind to its receptor within the cytosol or within the nucleus. In either case, this binding generates a hormone-receptor complex that moves toward the chromatin in the cell nucleus and binds to a particular segment of the cell&rsquos DNA. In contrast, thyroid hormones bind to receptors already bound to DNA. For both steroid and thyroid hormones, binding of the hormone-receptor complex with DNA triggers transcription of a target gene to mRNA, which moves to the cytosol and directs protein synthesis by ribosomes.
Figure 2: A steroid hormone directly initiates the production of proteins within a target cell. Steroid hormones easily diffuse through the cell membrane. The hormone binds to its receptor in the cytosol, forming a receptor&ndashhormone complex. The receptor&ndashhormone complex then enters the nucleus and binds to the target gene on the DNA. Transcription of the gene creates a messenger RNA that is translated into the desired protein within the cytoplasm.
Pathways Involving Cell Membrane Hormone Receptors
Hydrophilic, or water-soluble, hormones are unable to diffuse through the lipid bilayer of the cell membrane and must therefore pass on their message to a receptor located at the surface of the cell. Except for thyroid hormones, which are lipid-soluble, all amino acid&ndashderived hormones bind to cell membrane receptors that are located, at least in part, on the extracellular surface of the cell membrane. Therefore, they do not directly affect the transcription of target genes, but instead initiate a signaling cascade that is carried out by a molecule called a second messenger . In this case, the hormone is called a first messenger .
The second messenger used by most hormones is cyclic adenosine monophosphate (cAMP) . In the cAMP second messenger system, a water-soluble hormone binds to its receptor in the cell membrane (Step 1 in Figure 3). This receptor is associated with an intracellular component called a G protein , and binding of the hormone activates the G-protein component (Step 2). The activated G protein in turn activates an enzyme called adenylyl cyclase , also known as adenylate cyclase (Step 3), which converts adenosine triphosphate (ATP) to cAMP (Step 4). As the second messenger, cAMP activates a type of enzyme called a protein kinase that is present in the cytosol (Step 5). Activated protein kinases initiate a phosphorylation cascade , in which multiple protein kinases phosphorylate (add a phosphate group to) numerous and various cellular proteins, including other enzymes (Step 6).
Figure 3: Water-soluble hormones cannot diffuse through the cell membrane. These hormones must bind to a surface cell-membrane receptor. The receptor then initiates a cell-signaling pathway within the cell involving G proteins, adenylyl cyclase, the secondary messenger cyclic AMP (cAMP), and protein kinases. In the final step, these protein kinases phosphorylate proteins in the cytoplasm. This activates proteins in the cell that carry out the changes specified by the hormone.
The phosphorylation of cellular proteins can trigger a wide variety of effects, from nutrient metabolism to the synthesis of different hormones and other products. The effects vary according to the type of target cell, the G proteins and kinases involved, and the phosphorylation of proteins. Examples of hormones that use cAMP as a second messenger include calcitonin, which is important for bone construction and regulating blood calcium levels glucagon, which plays a role in blood glucose levels and thyroid-stimulating hormone, which causes the release of T3 and T4 from the thyroid gland.
Overall, the phosphorylation cascade significantly increases the efficiency, speed, and specificity of the hormonal response, as thousands of signaling events can be initiated simultaneously in response to a very low concentration of hormone in the bloodstream. However, the duration of the hormone signal is short, as cAMP is quickly deactivated by the enzyme phosphodiesterase (PDE) , which is located in the cytosol. The action of PDE helps to ensure that a target cell&rsquos response ceases quickly unless new hormones arrive at the cell membrane.
Importantly, there are also G proteins that decrease the levels of cAMP in the cell in response to hormone binding. For example, when growth hormone&ndashinhibiting hormone (GHIH), also known as somatostatin, binds to its receptors in the pituitary gland, the level of cAMP decreases, thereby inhibiting the secretion of human growth hormone.
Not all water-soluble hormones initiate the cAMP second messenger system. One common alternative system uses calcium ions as a second messenger. In this system, G proteins activate the enzyme phospholipase C (PLC), which functions similarly to adenylyl cyclase. Once activated, PLC cleaves a membrane-bound phospholipid into two molecules: diacylglycerol (DAG) and inositol triphosphate (IP3) . Like cAMP, DAG activates protein kinases that initiate a phosphorylation cascade. At the same time, IP3 causes calcium ions to be released from storage sites within the cytosol, such as from within the smooth endoplasmic reticulum. The calcium ions then act as second messengers in two ways: they can influence enzymatic and other cellular activities directly, or they can bind to calcium-binding proteins, the most common of which is calmodulin. Upon binding calcium, calmodulin is able to modulate protein kinase within the cell. Examples of hormones that use calcium ions as a second messenger system include angiotensin II, which helps regulate blood pressure through vasoconstriction, and growth hormone&ndashreleasing hormone (GHRH), which causes the pituitary gland to release growth hormones.
Factors Affecting Target Cell Response
You will recall that target cells must have receptors specific to a given hormone if that hormone is to trigger a response. But several other factors influence the target cell response. For example, the presence of a significant level of a hormone circulating in the bloodstream can cause its target cells to decrease their number of receptors for that hormone. This process is called downregulation , and it allows cells to become less reactive to the excessive hormone levels. When the level of a hormone is chronically reduced, target cells engage in upregulation to increase their number of receptors. This process allows cells to be more sensitive to the hormone that is present. Cells can also alter the sensitivity of the receptors themselves to various hormones.
Two or more hormones can interact to affect the response of cells in a variety of ways. The three most common types of interaction are as follows:
- The permissive effect, in which the presence of one hormone enables another hormone to act. For example, thyroid hormones have complex permissive relationships with certain reproductive hormones. A dietary deficiency of iodine, a component of thyroid hormones, can therefore affect reproductive system development and functioning.
- The synergistic effect, in which two hormones with similar effects produce an amplified response. In some cases, two hormones are required for an adequate response. For example, two different reproductive hormones&mdashFSH from the pituitary gland and estrogens from the ovaries&mdashare required for the maturation of female ova (egg cells).
- The antagonistic effect, in which two hormones have opposing effects. A familiar example is the effect of two pancreatic hormones, insulin and glucagon. Insulin increases the liver&rsquos storage of glucose as glycogen, decreasing blood glucose, whereas glucagon stimulates the breakdown of glycogen stores, increasing blood glucose.
Regulation of Hormone Secretion
To prevent abnormal hormone levels and a potential disease state, hormone levels must be tightly controlled. The body maintains this control by balancing hormone production and degradation. Feedback loops govern the initiation and maintenance of most hormone secretion in response to various stimuli.
Role of Feedback Loops
The contribution of feedback loops to homeostasis will only be briefly reviewed here. Positive feedback loops are characterized by the release of additional hormone in response to an original hormone release. The release of oxytocin during childbirth is a positive feedback loop. The initial release of oxytocin begins to signal the uterine muscles to contract, which pushes the fetus toward the cervix, causing it to stretch. This, in turn, signals the pituitary gland to release more oxytocin, causing labor contractions to intensify. The release of oxytocin decreases after the birth of the child.
The more common method of hormone regulation is the negative feedback loop. Negative feedback is characterized by the inhibition of further secretion of a hormone in response to adequate levels of that hormone. This allows blood levels of the hormone to be regulated within a narrow range. An example of a negative feedback loop is the release of glucocorticoid hormones from the adrenal glands, as directed by the hypothalamus and pituitary gland. As glucocorticoid concentrations in the blood rise, the hypothalamus and pituitary gland reduce their signaling to the adrenal glands to prevent additional glucocorticoid secretion (Figure 4).
Figure 4: The release of adrenal glucocorticoids is stimulated by the release of hormones from the hypothalamus and pituitary gland. This signaling is inhibited when glucocorticoid levels become elevated by causing negative signals to the pituitary gland and hypothalamus.
Role of Endocrine Gland Stimuli
Reflexes triggered by both chemical and neural stimuli control endocrine activity. These reflexes may be simple, involving only one hormone response, or they may be more complex and involve many hormones, as is the case with the hypothalamic control of various anterior pituitary&ndashcontrolled hormones.
Humoral stimuli are changes in blood levels of non-hormone chemicals, such as nutrients or ions, which cause the release or inhibition of a hormone to, in turn, maintain homeostasis. For example, osmoreceptors in the hypothalamus detect changes in blood osmolarity (the concentration of solutes in the blood plasma). If blood osmolarity is too high, meaning that the blood is not dilute enough, osmoreceptors signal the hypothalamus to release ADH. The hormone causes the kidneys to reabsorb more water and reduce the volume of urine produced. This reabsorption causes a reduction of the osmolarity of the blood, diluting the blood to the appropriate level. The regulation of blood glucose is another example. High blood glucose levels cause the release of insulin from the pancreas, which increases glucose uptake by cells and liver storage of glucose as glycogen.
An endocrine gland may also secrete a hormone in response to the presence of another hormone produced by a different endocrine gland. Such hormonal stimuli often involve the hypothalamus, which produces releasing and inhibiting hormones that control the secretion of a variety of pituitary hormones.
In addition to these chemical signals, hormones can also be released in response to neural stimuli. A common example of neural stimuli is the activation of the fight-or-flight response by the sympathetic nervous system. When an individual perceives danger, sympathetic neurons signal the adrenal glands to secrete norepinephrine and epinephrine. The two hormones dilate blood vessels, increase the heart and respiratory rate, and suppress the digestive and immune systems. These responses boost the body&rsquos transport of oxygen to the brain and muscles, thereby improving the body&rsquos ability to fight or flee.
Bisphenol A and Endocrine Disruption
You may have heard news reports about the effects of a chemical called bisphenol A (BPA) in various types of food packaging. BPA is used in the manufacturing of hard plastics and epoxy resins. Common food-related items that may contain BPA include the lining of aluminum cans, plastic food-storage containers, drinking cups, as well as baby bottles and &ldquosippy&rdquo cups. Other uses of BPA include medical equipment, dental fillings, and the lining of water pipes.
Research suggests that BPA is an endocrine disruptor, meaning that it negatively interferes with the endocrine system, particularly during the prenatal and postnatal development period. In particular, BPA mimics the hormonal effects of estrogens and has the opposite effect&mdashthat of androgens. The U.S. Food and Drug Administration (FDA) notes in their statement about BPA safety that although traditional toxicology studies have supported the safety of low levels of exposure to BPA, recent studies using novel approaches to test for subtle effects have led to some concern about the potential effects of BPA on the brain, behavior, and prostate gland in fetuses, infants, and young children. The FDA is currently facilitating decreased use of BPA in food-related materials. Many US companies have voluntarily removed BPA from baby bottles, &ldquosippy&rdquo cups, and the linings of infant formula cans, and most plastic reusable water bottles sold today boast that they are &ldquoBPA free.&rdquo In contrast, both Canada and the European Union have completely banned the use of BPA in baby products.
The potential harmful effects of BPA have been studied in both animal models and humans and include a large variety of health effects, such as developmental delay and disease. For example, prenatal exposure to BPA during the first trimester of human pregnancy may be associated with wheezing and aggressive behavior during childhood. Adults exposed to high levels of BPA may experience altered thyroid signaling and male sexual dysfunction. BPA exposure during the prenatal or postnatal period of development in animal models has been observed to cause neurological delays, changes in brain structure and function, sexual dysfunction, asthma, and increased risk for multiple cancers. In vitro studies have also shown that BPA exposure causes molecular changes that initiate the development of cancers of the breast, prostate, and brain. Although these studies have implicated BPA in numerous ill health effects, some experts caution that some of these studies may be flawed and that more research needs to be done. In the meantime, the FDA recommends that consumers take precautions to limit their exposure to BPA. In addition to purchasing foods in packaging free of BPA, consumers should avoid carrying or storing foods or liquids in bottles with the recycling code 3 or 7. Foods and liquids should not be microwave-heated in any form of plastic: use paper, glass, or ceramics instead.
Hormones are derived from amino acids or lipids. Amine hormones originate from the amino acids tryptophan or tyrosine. Larger amino acid hormones include peptides and protein hormones. Steroid hormones are derived from cholesterol.
Steroid hormones and thyroid hormone are lipid soluble. All other amino acid&ndashderived hormones are water soluble. Hydrophobic hormones are able to diffuse through the membrane and interact with an intracellular receptor. In contrast, hydrophilic hormones must interact with cell membrane receptors. These are typically associated with a G protein, which becomes activated when the hormone binds the receptor. This initiates a signaling cascade that involves a second messenger, such as cyclic adenosine monophosphate (cAMP). Second messenger systems greatly amplify the hormone signal, creating a broader, more efficient, and faster response.
Hormones are released upon stimulation that is of either chemical or neural origin. Regulation of hormone release is primarily achieved through negative feedback. Various stimuli may cause the release of hormones, but there are three major types. Humoral stimuli are changes in ion or nutrient levels in the blood. Hormonal stimuli are changes in hormone levels that initiate or inhibit the secretion of another hormone. Finally, a neural stimulus occurs when a nerve impulse prompts the secretion or inhibition of a hormone.