Prostaglandin-mediated Mechanism Research Articles

Alcohol reinstatement after prolonged abstinence from alcohol drinking by female adolescent rats: Roles of cyclooxygenase-2 and the prostaglandin E2 receptor 1

BackgroundAdolescent alcohol misuse is a global problem that can significantly increase the reinstatement of alcohol drinking during re-exposure after abstinence, but the mechanism that causes this increase is unknown. Female adolescents are an understudied population but they are particularly vulnerable to adolescent-onset alcohol abuse. We aimed to determine how adolescent-onset alcohol drinking affects pro-inflammatory mediators endothelin-1 (ET-1), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the brain and the role of COX-2 and PGE2 in EtOH reinstatement in adolescent females. MethodsAdolescent female rats were exposed to a 2-bottle choice paradigm of water vs 5% ethanol (EtOH) every other day over a 21 day period. ET-1 and COX-2 proteins were measured in the dorsal striatum (DS) after a 4 week abstinence from EtOH drinking. The COX-2 inhibitor nimesulide was then administered during abstinence prior to an EtOH reinstatement or sucrose preference or to measure PGE2 content. The PGE2 receptor 1 (EP1) antagonist SC-51089 was then administered prior to EtOH reinstatement during which EtOH intake was measured. ResultsEtOH drinking significantly increased ET-1 by 33.8 ± 8.9% and COX-2 by 71.4 ± 24.3% in the DS. Treatment with nimesulide during abstinence attenuated EtOH intake during reinstatement after prolonged abstinence by 40.3 ± 12.4% compared to saline controls. Adolescent EtOH drinking and abstinence increased PGE2 150.5 ± 30.9% in the DS and nimesulide attenuated this increase. SC-51089 treatment during abstinence attenuated EtOH reinstatement by 48.1 ± 8.4% compared to DMSO controls. ConclusionsThese experiments identified a prostaglandin-mediated mechanism that offers a putative pharmacological target to attenuate EtOH reinstatement after adolescent-onset EtOH drinking.

Food and water intake, body temperature and metabolic consequences of interleukin-1β microinjection into the cingulate cortex of the rat

In order to elucidate whether cytokine mechanisms of the cingulate cortex (cctx) are important in the central regulation of homeostasis, in the present study, feeding-metabolic effects of direct bilateral microinjection of interleukin-1β (IL-1β) into the cctx of the rat have been investigated. Short- (2h), medium (12h) and long-term (24h) food and water intakes and body temperature were measured after the intracerebral administration of this primary cytokine or vehicle solution, with or without paracetamol pretreatment. The effect of IL-1β on the blood glucose level of animals was examined in glucose tolerance test (GTT), and concentrations of relevant plasma metabolites (total cholesterol, HDL, LDH, triglycerides, uric acid) were additionally also determined following the above microinjections. In contrast to causing no major alteration in the food and water intakes, the cytokine treatment evoked significant increase in the body temperature of the rats. Prostaglandin-mediated mechanisms were shown to have important role in the mode of this action of IL-1β, since paracetamol pretreatment partially prevented the development of the above mentioned hyperthermia. In the GTT, no considerable difference was observed between the blood glucose levels of the cytokine treated and control animals. Following IL-1β microinjection, however, significant decrease of HDL and total cholesterol was found. Our present findings indicate that elucidating the IL-1β mediated homeostatic control mechanisms in the cingulate cortex may lead to the better understanding not only the regulatory entities of the healthy organism but also those found in obesity, diabetes mellitus and other worldwide rapidly spreading feeding-metabolic disorders.

Acute central effects of alarin on the regulation on energy homeostasis

Hypothalamic neuropeptides influence the main components of energy balance: metabolic rate, food intake, body weight as well as body temperature, by exerting either an overall anabolic or catabolic effect. The contribution of alarin, the most recently discovered member of the galanin peptide family to the regulation of energy metabolism has been suggested. Our aim was to analyze the complex thermoregulatory and food intake-related effects of alarin in rats.Adult male Wistar rats received different doses of alarin (0.3; 1; 3 and 15μg corresponding approximately to 0.1, 0.33, 1, and 5 nmol, respectively) intracerebroventricularly. Regarding thermoregulatory analysis, oxygen consumption (indicating metabolic rate), core temperature and heat loss (assessed by tail skin temperature) were recorded in an Oxymax indirect calorimeter system complemented with thermocouples and Benchtop thermometer. In order to investigate potential prostaglandin-mediated mechanisms of the hyperthermic effect of alarin, effects of intraperitoneally applied non-selective (indomethacin, 2mg/kg) or selective cyclooxygenase inhibitor (COX-2 inhibitor meloxicam, 1; 2mg/kg) were tested. Effects of alarin on daytime and nighttime spontaneous food intake, as well as, 24-h fasting-induced re-feeding were recorded in an automated FeedScale system.Alarin increased oxygen consumption with simultaneous suppression of heat loss leading to a slow coordinated rise in core temperature. Both applied COX-inhibitors suppressed this action. Alarin failed to induce daytime food intake, but suppressed spontaneous nighttime and also fasting-induced re-feeding food intake.Alarin appears to elicit a slow anorexigenic and prostaglandin-mediated, fever-like hyperthermic response in rats. Such a combination would characterize a catabolic mediator. The potential involvement of alarin in sickness behavior may be assumed.

Antiulcer and antiproliferative properties of spent brewer's yeast peptide extracts for incorporation into foods.

The main objective was to study the antiulcer and antiproliferative potential of yeast peptide extract for further incorporation into functional foods. Peptide concentrates were obtained by hydrolysis of spent brewer's yeast proteins followed by a filtration process. In order to prove the possible protection of gastric mucosa, an animal model with ulcerative lesions caused by oral administration of absolute ethanol was used. The peptide fraction <3 kDa was able to reduce gastric injuries to significant levels (p < 0.001) and the effective dose (DE50) was 816 mg per kg bw. The cytoprotective effect appears to depend on a prostaglandin-mediated mechanism and also on a nonspecific mechanism. The antiproliferative activity of the extract in nine different human tumoral cell lines was tested. The results exhibited a promising antiproliferative activity against the cell line K-562 (leukemia). The results suggest that a new peptide extract can be used to develop new value-added functional food products, although further studies are required.

In vivo mechanical loading rapidly activates β-catenin signaling in osteocytes through a prostaglandin mediated mechanism

The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo.

Tumor necrosis factor alpha inhibits in vitro bovine embryo development through a prostaglandin mediated mechanism

Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα) are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage (17.5 ± 2.4%, P < 0.01) compared with controls (30.5 ± 2.4%) or embryos developed in TNFα plus indomethacin (25.8 ± 2.8%). There was no difference between control embryos and embryos developed in TNFα plus indomethacin. These results indicate that TNFα is inhibitory to the in vitro development of bovine embryos and that this inhibition may be mediated by prostaglandins because it can be blocked by indomethacin.

Ageing uncompensated: exercise, nitric oxide and hypoxia

Ageing uncompensated: exercise, nitric oxide and hypoxia

Prolonged ischaemia impairs muscle blood flow and oxygen uptake dynamics during subsequent heavy exercise

Muscle oxygen uptake ( ˙VO₂,mus) dynamics at the onset of exercise can be affected by prior heavy exercise.We tested the hypothesis that elevated forearm blood flow (FBF) following prior circulatory occlusion would also be associated with accelerated ˙VO₂,mus dynamics during subsequent heavy hand-grip exercise. Ten trained young men performed 5 min of heavy hand-grip exercise at 30% MVC as a control (CON), and four additional heavy bouts after brief recovery from: (1) prior heavy exercise (Heavy A), (2) heavy exercise followed by 2 min occlusion (Heavy B), (3) 15 min occlusion (Heavy C), and (4) 5 min occlusion with 1 min of moderate exercise during occlusion (Heavy D). FBF was measured by ultrasound and arterial venous oxygen content difference was calculated from venous blood samples to estimate ˙VO₂,mus. FBF and ˙VO₂,mus dynamics were quantified from the rise time. All priming conditions elevated FBF immediately before the start of subsequent heavy bout (Heavy A: 207.4 ±92.8, B: 207.8±75.8, C: 135.8±59.2, D: 199.5±59.0 vs. CON: 57.4±16.6mlmin−1, P <0.01). Unexpectedly, prior occlusion reduced FBF and O2 extraction at the onset of subsequent heavy exercise and consequently slowed ˙VO₂,mus dynamics (Heavy C: rise time=95.9±28.9 vs. CON: 58.6±14.3 s, P <0.01). FBF and ˙VO₂,mus dynamics were faster in Heavy A, B and D compared to CON (P <0.05). Overall, there was a positive correlation between the rise times for ˙VO₂,mus and FBF (r² =0.75) indicating that ˙VO₂,mus dynamics during heavy forearm exercise are linked to O₂ delivery in trained young men. To investigate a possible mechanism for slower adaptation of ˙VO₂,mus following ischaemia, the prior occlusion condition was repeated after ingesting a high dose of ibuprofen. This resulted in restoration of the FBF and ˙VO₂,mus to control levels suggesting that a prostaglandin-mediated mechanism after occlusion retarded the adaptation of blood flow and oxygen consumption at the onset of subsequent heavy exercise.

Rutin-induced endothelium-dependent vasorelaxation in rat aortic Rings and the underlying mechanism.

The aim of the present study was to determine the possible mechanism by which rutin causes vasodilatation in isolated thoracic aorta rings from the rat. The effects of rutin on rings preconstricted with phenylephrine, with or without endothelium, were determined using an organ bath technique. The mechanism was explored by measuring the effects of the nitric oxide synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), the guanylyl cyclase inhibitor methylene blue, the cyclooxygenase inhibitor indomethacin, the ATP-sensitive K+channel blocker glibenclamide and the beta-adrenoceptor antagonist propranolol. Rutin at the range of 10-160 mumol/L caused dose-dependent vasorelaxation in preconstricted endothelium-intact rings, but had no effect on rings without endothelium. The maximal response calculated from the vasorelaxation curves of rutin was 44.28 plus or mines 7.48%. Pretreatment with L-NAME (0.1 mmol/L), methylene blue (10 mumol/L), glibenclamide (10 mmol/L) or indomethacin (10 mmol/L) attenuated the vasorelaxation induced by rutin in endothelium-intact rings. Glibenclamide (10 mmol/L) enhanced the vasorelaxation of rutin. Propranolol (10 mumol/L) did not block the effect of rutin. The results indicate that vasorelaxation is induced by rutin via the nitric oxide-guanylyl cyclase pathway and a prostaglandin-mediated mechanism, as well as activation of the ATP-sensitive potassium channel.

IL-1 beta depresses respiration and anoxic survival via a prostaglandin-dependent pathway in neonatal rats.

IL-1 beta has been proposed to be an important mediator linking infection, apnea, and sudden infant death syndrome. We hypothesized that IL-1 beta acts in this capacity by depressing brainstem respiratory neurons via a prostaglandin-dependent pathway. For studying the effects of IL-1 beta on respiration as well as the mechanism underlying its actions, 7-d-old rats received an initial injection (i.p.) of NaCl or a cyclooxygenase inhibitor (indomethacin, 10 mg/kg) followed by a second injection (i.p.) at 30 min of NaCl, recombinant rat IL-1 beta (10 microg/kg), or lipopolysaccharide (LPS; 100 microg/kg). Respiration during normoxia and in response to anoxia (100% N2) was examined at 60 min after the second injection using flow and barometric plethysmography. Animals given IL-1 beta breathed more slowly and died more often after anoxia. LPS also reduced the rats' ability to autoresuscitate and survive an anoxic challenge. Indomethacin prevented the depressive effects during normoxia and the adverse effects on survival. For investigating drug-induced changes in central respiratory activity, IL- 1 beta (1.0 or 1.25 ng/mL) and prostaglandin E2 (5 or 20 microg/L) was applied to the brainstem-spinal cord preparation of 0- to 4-d-old rats. Whereas IL-1 beta exerted no effect on respiration measured at the C4 ventral root during a 60-min period, prostaglandin E2 reversibly inhibited respiratory activity. These findings suggest that IL-1 beta does not inhibit respiratory neurons directly but may depress breathing and hypoxic defense via a prostaglandin-mediated mechanism.

Centrally applied nitric oxide donor elevates plasma corticosterone by activation of the hypothalamic noradrenergic neurons in rats

The present study was undertaken to investigate the mechanisms involved in a nitric oxide donor [3-morpholino-sydnonimine (SIN-1)]-induced activation of hypothalamic–pituitary–adrenal axis in urethane- and α-chloralose-anesthetized rats. Intracerebroventricularly (i.c.v.) administered SIN-1 (250 and 500 μg/animal) effectively and dose-dependently elevated plasma levels of corticosterone. Pretreatment with phentolamine (250 μg/animal, i.c.v.), an α-adrenoceptor antagonist, attenuated the elevation of plasma corticosterone evoked by SIN-1, but sotalol (300 μg/animal, i.c.v.), a β-adrenoceptor antagonist, was without effects. The same doses of SIN-1 also increased the release of noradrenaline in the hypothalamic paraventricular nucleus (PVN) measuring microdialysis technique, and this increase was abolished by tetrodotoxin (1 μM) administered into the perfusion solution of the PVN. Furthermore, pretreatment with indomethacin (500 μg/animal, i.c.v.), a cyclooxygenase inhibitor, abolished the SIN-1-induced elevations of both noradrenaline in the PVN and plasma corticosterone. These results suggest that i.c.v. administered SIN-1 activates central noradrenergic neurons innervating the PVN by prostaglandin-mediated mechanisms. Released noradrenaline in the PVN elevates plasma levels of corticosterone via an activation of the central α-adrenoreceptors in rats.

Activation of prostanoid EP(3) and EP(4) receptor mRNA-expressing neurons in the rat parabrachial nucleus by intravenous injection of bacterial wall lipopolysaccharide.

Systemic inflammation activates central autonomic circuits, such as neurons in the pontine parabrachial nucleus. This activation may be the result of afferent signaling through the vagus nerve, but it may also depend on central prostaglandin-mediated mechanisms. Recently, we have shown that neurons in the parts of the parabrachial nucleus that are activated by immune challenge express prostaglandin receptors of the EP(3) and EP(4) subtypes, but it remains to be determined if the prostaglandin receptor-expressing neurons are identical to those that respond to immune stimuli. In the present study, bacterial wall lipopolysaccharide was injected intravenously in adult male rats and the expression of c-fos mRNA and of EP(3) and EP(4) receptor mRNA was examined with complementary RNA probes labeled with digoxigenin and radioisotopes, respectively. Large numbers of neurons in the external lateral parabrachial subnucleus, a major target of vagal-solitary tract efferents, expressed c-fos mRNA. Quantitative analysis showed that about 60% (range 40%-79%) of these neurons also expressed EP(3) receptor mRNA. Conversely, slightly more than 50% (range 48%-63%) of the EP(3) receptor-expressing neurons in the same subnucleus coexpressed c-fos mRNA. In contrast, few EP(4) receptor-expressing neurons were c-fos positive, with the exception of a small population located in the superior lateral and dorsal lateral subnuclei. These findings show that immune challenge activates central autonomic neurons that could be the target of centrally produced prostaglandin E(2), suggesting that synaptic signaling and paracrine mechanisms may interact on these neurons.

The contractile action of platelet-activating factor on gallbladder smooth muscle.

Platelet-activating factor (PAF) may be a mediator of some sequelae of cholecystitis, a disorder with gallbladder motor dysfunction. The aims of this study were to determine the effect and mechanism of PAF on gallbladder muscle. Exogenous administration of PAF-16 or PAF-18 caused dose-dependent contractions of gallbladder muscle strips in vitro with threshold doses of 1 ng/ml and 10 ng/ml, respectively. The PAF-induced contractions were not significantly reduced by TTX, atropine, or hexamethonium but were significantly inhibited with the PAF receptor antagonists ginkolide B and CV-3988. The PAF-induced contraction was reduced by indomethacin. Preventing influx of extracellular calcium with a calcium-free solution nearly abolished the PAF contractile response. Nifedipine inhibited the PAF contractile response, whereas ryanodine had no effect. Pertussis toxin reduced the PAF contractile response. In conclusion, PAF causes gallbladder contraction through specific PAF receptors on gallbladder muscle. These PAF receptors appear to be linked to a prostaglandin-mediated mechanism and to pertussis toxin-sensitive G proteins. The contractile response is largely mediated through the utilization of extracellular calcium influx through voltage-dependent calcium channels.

Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism

Background & Aims: The bone marrow and the intestine are the major sites of radiation-induced injury. The cellular response to radiation injury in the intestine or bone marrow can be modulated by agents given before irradiation. Lipopolysaccharide is known to be radioprotective in the bone marrow, but its effect on the intestine is not known. We sought to determine if lipopolysaccharide is radioprotective in the intestine and, if so, to determine the mechanism of its radioprotective effects. Methods: Mice were treated with parenteral lipopolysaccharide or vehicle and then irradiated (14 Gy total body irradiation in a cesium irradiator). The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. Results: Parenteral administration of lipopolysaccharide 2-24 hours before irradiation resulted in a 2-fold increase in the number of surviving crypts 3.5 days after irradiation. The radioprotective effects of lipopolysaccharide could be eliminated by coadministration of a selective inhibitor of cyclooxygenase 2. Lipopolysaccharide was radioprotective in wild-type mice but not in mice with a disrupted cyclooxygenase 2. Parenteral administration of lipopolysaccharide resulted in increased production of prostaglandins in the intestine and in the induction of cyclooxygenase 2 expression in subepithelial fibroblasts and in villous, but not crypt, epithelial cells. Conclusions Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-dependent pathway. GASTROENTROLOGY 2000;118:1106-1116

Pharmacological characterization of the cardiovascular responses elicited by kinin B(1) and B(2) receptor agonists in the spinal cord of streptozotocin-diabetic rats.

Kinin receptor agonists and antagonists at the B(1) and B(2) receptors were injected intrathecally (i.t., at T-9 spinal cord level) to conscious unrestrained rats and their effects on mean arterial pressure (MAP) and heart rate (HR) were compared in streptozotocin (STZ)-diabetic rats (65 mg kg(-1) STZ, i.p. 3 weeks earlier) and aged-matched control rats. The B(1) receptor agonist, des-Arg(9)-Bradykinin (BK) (3.2 - 32.5 nmol), evoked dose-dependent increases in MAP and tachycardia during the first 10 min post-injection in STZ-diabetic rats only. The cardiovascular response to 6.5 nmol des-Arg(9)-BK was reversibly blocked by the prior i.t. injection of antagonists for the B(1) receptor ([des-Arg(10)]-Hoe 140, 650 pmol or [Leu(8)]-des-Arg(9)-BK, 65 nmol) and B(2) receptor (Hoe 140, 81 pmol or FR173657, 81 pmol) or by indomethacin (5 mg kg(-1), i.a.). The i.t. injection of BK (8.1 - 810 pmol) induced dose-dependent increases in MAP which were accompanied either by tachycardiac (STZ-diabetic rats) or bradycardiac (control rats) responses. The pressor response to BK was significantly greater in STZ-diabetic rats. The cardiovascular response to 81 pmol BK was reversibly blocked by 81 pmol Hoe 140 or 81 pmol FR173657 but not by B(1) receptor antagonists nor by indomethacin in STZ-diabetic rats. The data suggest that the activation of kinin B(1) receptor in the spinal cord of STZ-diabetic rats leads to cardiovascular changes through a prostaglandin mediated mechanism. Thus, this study affords an accessible model for studying the expression, the pharmacology and physiopathology of the B(1) receptor in the central nervous system.

Tissue prostaglandin levels in familial adenomatous polyposis patients treated with sulindac

Recent work has demonstrated a correlation between frequency of aspirin ingestion and colorectal cancer prevention. Sulindac, another nonsteroidal anti-inflammatory drug (NSAID), has been shown to cause polyp regression and a fall in cell proliferation in patients with familial adenomatous polyposis, who are destined to develop colorectal cancer unless the colon is removed. However, the mode of action of NSAIDs in colorectal carcinogenesis prevention remains to be determined, although a prostaglandin-mediated mechanism seems likely. Rectal or duodenal biopsies from 20 patients with familial adenomatous polyposis, who had been randomized to sulindac or placebo, were analyzed for prostaglandin (PG) E2 and F2 alpha levels before and after treatment. A significant fall in prostaglandin E2 and F2 alpha levels was seen in patients who were on sulindac; this correlated with a visual improvement in number and size of polyps in the same patients (P = 0.0096; PGE2, P = 0.036; PGF2 alpha, Spearman's rank correlation). Nonsteroidal anti-inflammatory drugs may prevent colorectal cancer by their inhibition of prostaglandin synthesis. Prostaglandins may be implicated in carcinogenesis through an increase in cell proliferation, through immunosuppression, by increasing neovascularization, or via a mutagenic effect.

Indomethacin inhibition of ossification induced by direct current stimulation

In this study, we sought to clarify the mechanism of ossification induced by direct current stimulation by analyzing changes in local blood flow and vascular permeability and by examining the involvement of chemical mediators. Changes in blood flow were studied with laser Doppler flowmetry, and vascular permeability was determined by microquantification. To examine the involvement of chemical mediators, we determined the effect on vascular permeability of histamine H1 and H2-receptor blockers and indomethacin. In addition, direct current stimulation was performed during administration of indomethacin to determine whether indomethacin inhibits electrically induced callus formation. Local blood flow remained unchanged in the control group and in the group receiving 5 microA of stimulation, but it increased in the groups receiving 10 and 50 microA. Vascular permeability increased in the 5 and 10 microA stimulation groups. This increase was not suppressed by histamine-receptor blockers, but it was suppressed by indomethacin. Two weeks of electrical stimulation without concomitant indomethacin treatment resulted in active callus formation around the needle electrode and in the vicinity of the endosteum; however, direct current stimulation during administration of indomethacin inhibited callus formation. The results suggest that these changes serve as microenvironmental factors that play an important role in the promotion of ossification and that a prostaglandin-mediated mechanism is involved in the promotion of ossification by direct current stimulation.

Adaptive cytoprotection of the rat duodenum is not dependent on nitric oxide-induced changes in blood flow.

Mild irritation of the rat duodenum enhances mucosal resistance to acid via a prostaglandin-mediated mechanism. Prostaglandins increase mucosal blood flow, but it is not known whether such changes in blood flow significantly contribute to adaptive cytoprotection. We induced blood flow changes by manipulating the arginine-nitric oxide pathway, which is independent from prostaglandin synthesis, and determined the resulting effects on the cytoprotective response. In anesthetized rats, we tested the effects of intraduodenal infusion of mild acid on duodenal blood flow and on the prevention of mucosal damage by subsequent strong acid in control, indomethacin-pretreated, and NG-nitro-L-arginine-pretreated rats (NG-nitro-L-arginine inhibits the nitric oxide synthase). Additional experiments tested the effects of the prostaglandin analogue 16,16-dimethyl-prostaglandin (PG) E2 or sodium nitroprusside (nitric oxide donor) on duodenal blood flow and on mucosal protection against acid. Exposure of the duodenal mucosa to mild acid increased duodenal blood flow and mucosal resistance to acid. Instillation of 16,16-dimethyl-PGE2 also increased blood flow and mucosal resistance to acid. However, sodium nitroprusside increased blood flow without increasing mucosal resistance to acid, and NG-nitro-L-arginine inhibited the change in blood flow induced by acid while preserving the adaptive cytoprotection phenomenon intact. In conclusion, adaptive cytoprotection of the duodenal mucosa appears to be independent of changes in blood flow.

Effects of platelet-derived growth factor isoforms on calcium release from neonatal mouse calvariae

Platelet-derived growth factor (PDGF) is the major growth factor in serum for cells of mesenchymal origin and induces many different activities, including bone resorption. Since the initial report that PDGF stimulated calcium release from bone organ cultures, it has been shown that PDGF is a dimeric protein consisting of two disulfide bonded polypeptides encoded by different genes. Three isoforms of the two gene products have been isolated. We compared the capacity of each isoform to stimulate calcium release from radiolabeled mouse calvariae. PDGF-AB from human platelets and recombinant PDGF'-BB isoforms significantly stimulated calcium release at 5 ng/ml, but not in lower doses. Recombinant PDGF-AA did not induce calcium release. Indomethacin blocked the stimulated bone resorption, suggesting a prostaglandin-mediated mechanism of action. PDGF-induced calcium release was compared to TGF-β1 in the organ culture system. Approximately a 10-fold greater concentration of PDGF-AB and PDGF-BB was required to achieve a similar degree of calcium release as found in TGF-β1 treated calvariae. Thus, TGF-β1, PDGF-AB, and PDGF-BB significantly stimulated calcium release from mouse calvariae. This response is specific in that PDGF-AA did not stimulate calcium release.

Human parathyroid hormone-related peptide-(107-111) does not inhibit bone resorption in neonatal mouse calvariae.

Recent analysis of the structure-function relationship of human PTH-related peptide (hPTHrP) has led to the discovery that its direct inhibitory activity on osteoclastic bone resorption resides fully in the 107-111 sequence of the peptide, as assessed by a bone resorption assay using isolated rat osteoclasts. Here we report that hPTHrP-(107-111) is inactive in neonatal mouse calvariae in culture. hPTHrP-(107-111), at doses of 10(-12)-10(-6) M and incubation periods up to 96 h, did not affect either basal or agonist-stimulated 45Ca release from prelabeled neonatal mouse calvariae, while salmon calcitonin was a potent and powerful inhibitor of both basal and stimulated 45Ca release from bone. Moreover, salmon calcitonin, but not hPTHrP-(107-111), inhibited the increase in osteoclast number in hPTHrP-(1-34)-treated bones. Furthermore, hPTHrP-(107-139) also failed to inhibit 45Ca release and the hPTHrP-(1-34)-induced increase in osteoclast number in this organ culture model when tested under conditions identical to those for hPTHrP-(107-111). The addition of indomethacin to hPTHrP-(107-111)- or hPTHrP-(107-139)-treated bones was without effect, excluding the possibility that the direct inhibitory activity of these peptides on osteoclasts is ablated by a prostaglandin-mediated mechanism. Although the mechanism underlying the apparent inability of the carboxyl-terminal PTHrP fragments to inhibit osteoclastic bone resorption in neonatal mouse calvariae is unknown, it may involve the complex microenvironment of osteoclasts in intact bone, which contains a large variety of cell types other than osteoclasts.