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The purpose of this study was to clarify the relationship between neuron cells and astrocyte cells in regulating glutamate toxicity on the 10th and 20th day in vitro. A mixed primary culture system from newborn rats that contain cerebral cortex neurons cells was employed to investigate the glutamate toxicity. All cultures were incubated with various glutamate concentrations, then viability tests and histological analyses were performed. The activities of glutamate transporters were determined by using in vitro voltammetry technique. Viable cell number was decreased significantly on the 10th day at 10(-7) M and at 10(-6) M glutamate applications, however, viable cell number was not decreased at 20th day. Astrocyte number was increased nearly six times on the 20th day as compared to the 10th day. The peak point of glutamate reuptake capacity was about 2 × 10(-4) M on the 10th day and 10(-3) M on the 20th day. According to our results, we suggested that astrocyte age was important to maintain neuronal survival against glutamate toxicity. Thus, we revealed activation or a trigger point of glutamate transporters on astrocytes due to time since more glutamate was taken up by astrocytes when glutamate transporters on the astrocyte were triggered with high exogenous glutamate concentrations. In conclusion, the present investigation is the first voltammetric study on the reuptake parameters of glutamate in vitro.

Background: Clinical studies indicate that patients with post-traumatic stress disorder (PTSD) frequently share comorbidity with numerous chronic pain conditions. However, the sustained effects of PTSD-like stress over time on visceral nociception and hyperalgesia have been rarely studied, and the underlying mechanisms of stress-induced modulation of visceral hyperalgesia remain elusive. The purpose of this study was to investigate the characterization of visceral nociception and hyperalgesia over time in rats exposed to PTSD-like stress, and to explore the potential role of protein kinase C gamma (PKCγ) in mediating visceral hyperalgesia following exposure to PTSD-like stress., Results: On day 1, the rats exposed to single-prolonged stress (SPS, an established animal model for PTSD) exhibited an analgesic response and its visceromotor response (VMR) to graded colorectal distention (CRD) at 40 and 60 mmHg was reduced compared with the control group (all P < 0.05). On day 6, the VMR returned to the baseline value. However, as early as 7 days after SPS, VMR dramatically increased compared with its baseline value and that in the controls (all P < 0.001) and this increase persisted for 28 days, with the peak on day 9. Abdominal withdrawal reflex (AWR) scores were higher in SPS rats than in controls on days 7, 9, 14, 21 and 28 (all P < 0.001). Intrathecal administration of GF109203X (an inhibitor of PKC gamma), attenuated the SPS-induced increase in both VMR and AWR scores on days 7, 14, 21 and 28 (all P < 0.05). PKCγ protein expression determined by immunofluorescence was reduced in the spinal cord within 3 days after the exposure to SPS (P < 0.01), which returned to normal levels between days 4 and 6, and significantly increased from day 7, and this increase was maintained on days 14, 21, and 28 (all P < 0.001), with the peak on day 9. In addition, Western blotting showed a consistent trend in the changes of PKCγ protein expression., Conclusions: The modified SPS alters visceral sensitivity to CRD, and contributes to the maintenance of visceral hyperalgesia, which is associated with enhanced PKCγ expression in the spinal cord. Functional blockade of the PKCγ receptors attenuates SPS-induced visceral hyperalgesia. Thus, the present study identifies a specific molecular mechanism for visceral hyperalgesia which may pave the way for novel therapeutic strategies for PTSD-like conditions.

Recent studies demonstrate that glial glutamate transporter-1 (GLT-1) upregulation attenuates visceral nociception. The present work further characterized the effect of ceftriaxone- (CTX-) mediated GLT-1 upregulation on visceral hyperalgesia. Intrathecal pretreatment with dihydrokainate, a selective GLT-1 antagonist, produced a reversal of the antinociceptive response to bladder distension produced by CTX. The hyperalgesic response to urinary bladder distension caused by intravesicular acrolein was also attenuated by CTX treatment as was the enhanced time spent licking of abdominal area due to intravesicular acrolein. Bladder inflammation via cyclophosphamide injections enhanced the nociceptive to bladder distension; cohorts administered CTX and concomitant cyclophosphamide showed reduced hyperalgesic response. Cyclophosphamide-induced bladder hyperalgesia correlated with a significant 22% increase in GluR1 AMPA receptor subunit expression in the membrane fraction of the lumbosacral spinal cord, which was attenuated by CTX coadministration. Finally, neonatal colon insult-induced hyperalgesia caused by intracolonic mustard oil (2%) administration at P9 and P11 was attenuated by CTX. These studies suggest that GLT-1 upregulation (1) attenuates the hyperalgesia caused by bladder irritation/inflammation or by neonatal colonic insult, (2) acts at a spinal site, and (3) may produce antinociceptive effects by attenuating GluR1 membrane trafficking. These findings support further consideration of this FDA-approved drug to treat chronic pelvic pain syndromes.

The lysosomal storage pathology in Mucopolysaccharidosis (MPS) IIIB manifests in cells of virtually all organs. However, it is the profound role of the neurological pathology that leads to morbidity and mortality in this disease, and has been the major challenge to developing therapies. To date, MPS IIIB neuropathologic and therapeutic studies have focused predominantly on changes in the central nervous system (CNS), especially in the brain, and little is known about the disease pathology in the peripheral nervous system (PNS). This study demonstrates characteristic lysosomal storage pathology in dorsal root ganglia affecting neurons, satellite cells (glia) and Schwann cells. Lysosomal storage lesions were also observed in the myoenteric plexus and submucosal plexus, involving enteric neurons with enteric glial activation. Further, MPS IIIB mice developed progressive impairments in sensory functions, with significantly reduced response to pain stimulation that became detectable at 4-5 months of age as the disease progressed. These data demonstrate that MPS IIIB neuropathology manifests not only in the entire CNS but also the PNS, likely affecting both afferent and efferent neural signal transduction. This study also suggests that therapeutic development for MPS IIIB may benefit from targeting the entire nervous system.

The effective management of chronic pain remains enigmatic. There is a paucity of effective mechanistically-based approaches employed. Chronic visceral pain is a particularly difficult subcategory to manage. Glutamate is the most predominant excitatory neurotransmitter and mediates many aspects of sensory function including acute and chronic pain. There is a growing literature describing the efficacy of physiologically dominant glutamate transporter GLT-1 up-regulation in attenuating chronic visceral and somatic nociception. Since glutamate is the major excitatory neurotransmitter released in the first central synapse of the pain-transmitting afferent neurons, augmentation of GLT-1 activity, which reduces extracellular levels of glutamate, may be an important target for pain management strategies. This review summarizes studies in our laboratory and others which highlight findings that GLT-1 up-regulation by transgenic, pharmacologic and viral transfection approaches attenuate a host of nociceptive responses emanating from visceral or somatic sources in animal models. The study also outlines the future work that will be required to ascertain the translational potential of this approach.

Glutamatergic pathways mediate transmission of pain. Strategies to reduce glutamatergic neurotransmission may have beneficial effects to mitigate nociception. Recent work revealed that overexpression of the astrocytic glutamate transporter (GLT-1) by transgenic or pharmacologic approaches produced a diminished visceral nociceptive response to colonic distension. The purpose of this study was to determine the effect of GLT-1 overexpression on the visceromotor response to bladder distension. Increased glutamate uptake activity produced by 1-wk ceftriaxone (CTX) treatment attenuated 60-64% the visceromotor response to graded bladder distension compared with vehicle-treated mice. One-hour pretreatment with selective GLT-1 antagonist dihydrokainate reversed the blunted visceromotor response to bladder distension produced by 1-wk CTX, suggesting that GLT-1 overexpression mediated the analgesic effect of CTX. Moreover, sensitization of the visceromotor response to bladder distension produced by local bladder irritation (acrolein) was also attenuated by 1-wk CTX treatment. A model of cross-organ sensitization of bladder visceromotor response to distension was next studied to determine whether increased expression of GLT-1 can mitigate colon to bladder sensitization. Intracolonic trinitrobenzene sulfonic acid (TNBS) administered 1 h before eliciting the visceromotor response to graded bladder distension produced a 75-138% increase in visceromotor response compared with animals receiving intracolonic vehicle. In marked contrast, animals treated with 1-wk CTX + intracolonic TNBS showed no enhanced visceromotor response compared with the 1-wk vehicle + intracolonic vehicle group. The study suggests that GLT-1 overexpression attenuates the visceromotor response to bladder distension and both local irritant-induced and cross-organ-sensitized visceromotor response to bladder distension.

Visceral hypersensitivity is the leading complaint of functional bowel disorders. Central sensitization mediated by glutamate receptor activation is implicated in pathophysiology of visceral pain. The glial glutamate transporter EAAT2 is the principal mediator of glutamate clearance to terminate glutamate-mediated responses. Transgenic mice overexpressing human EAAT2 (EAAT2 mice), which exhibited a twofold enhanced glutamate uptake, showed 39% less writhing response to intraperitoneal acetic acid than nontransgenic littermates. Moreover, EAAT2 transgenic mice showed a 53-64% reduction in visceromotor response (VMR) to colorectal distension (CRD) in assessments of the response to graded increase in pressures. Corroborating the involvement of enhanced glutamate uptake, wild-type mice treated for 1 wk with ceftriaxone, an EAAT2 expression activator, showed a 49-70% reduction in VMR to CRD. Moreover, systemic pretreatment with the selective EAAT2 transporter blocker dihydrokainate reversed the ceftriaxone-blunted nociceptive response to CRD. However, the enhanced VMR to CRD produced by intracolonic ethanol was not significantly attenuated by 1-wk ceftriaxone pretreatment. The data suggest that enhanced glutamate uptake provides protective effects against colonic distension-induced nociception and represents an exciting new mechanistic approach leading to better therapeutic options to visceral pain disorders.

An emerging theme in the study of the pathophysiology of chronic and persistent pain is the role of pro-oxidant substances. Reactive oxygen species (ROS) have been implicated in contributing to and/or maintaining conditions of chronic pain. Recent pre-clinical reports suggest that antioxidants are effective analgesics in neuropathic and inflammatory pain models. The present study extends this work by examining the effect of three antioxidants on tissue injury-induced nociception. C57BL6 mice (20-25 g) were pretreated with either phenyl-N-tert-butylnitrone (PBN; 50 mg/kg, i.p.), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy (TEMPOL; 200 or 50 mg/kg, i.p.), N-acetyl-L-cysteine (NAC; 200 or 100mg/kg, i.p.), or vehicle (0.5 ml/100 g), 5 min before intraplantar formalin (10%, 20 microl) injection. Nociceptive responding, indicated by licking or biting the affected hindlimb, was quantified for 30 min after formalin injection. Each drug was effective in attenuating two or more phases (acute, quiescent, and tonic) of the formalin response. To assess putative site of action, intrathecal TEMPOL (380 nmol/5 microl, i.t.) was given 5 min before intraplantar formalin. Intrathecal TEMPOL produced a 83% reduction in nociceptive responding in the tonic phase, but no significant attenuation of the acute phase response. To confirm that the antioxidant property of intrathecal TEMPOL was responsible for its analgesic effect on the formalin-induced pain response, intrathecal TEMPOL was coadministered with the free radical donor tert-butylhydroperoxide (tert-BuOOH). Tert-BuOOH coadminstration reversed the TEMPOL-induced analgesia in the tonic intraplantar formalin response reduction. The data suggest that pro-oxidant species may be important mediators of tissue injury-induced algesia in rodents, and that a spinal site of action is implicated in the tonic response.

Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission and for generation and maintenance of spinal states of pain hypersensitivity via activation of glutamate receptors. Understanding the regulation of synaptically and non-synaptically released glutamate associated with pathological pain is important in exploring novel molecular mechanisms and developing therapeutic strategies of pathological pain. The glutamate transporter system is the primary mechanism for the inactivation of synaptically released glutamate and the maintenance of glutamate homeostasis. Recent studies demonstrated that spinal glutamate transporter inhibition relieved pathological pain, suggesting that the spinal glutamate transporter might serve as a therapeutic target for treatment of pathological pain. However, the exact function of glutamate transporter in pathological pain is not completely understood. This report will review the evidence for the role of the spinal glutamate transporter during normal sensory transmission and pathological pain conditions and discuss potential mechanisms by which spinal glutamate transporter is involved in pathological pain.

Glutamate is a major excitatory neurotransmitter in primary afferent terminals and is critical for normal spinal excitatory synaptic transmission. However, little is known about the regulation of synaptically released glutamate in the spinal cord under physiologic conditions. The sodium-dependent, high-affinity glutamate transporters are the primary mechanism for the clearance of synaptically released glutamate. In the present study, we found that intrathecal injection of glutamate transporter blockers DL-threo-beta-benzyloxyaspartate (TBOA) and dihydrokainate produced significant and dose-dependent spontaneous nociceptive behaviors, such as licking, shaking, and caudally directed biting, phenomena similar to the behaviors caused by intrathecal glutamate receptor agonists. Intrathecal TBOA also led to remarkable hypersensitivity in response to thermal and mechanical stimuli. These behavioral responses could be significantly blocked by intrathecal injection of the NMDA receptor antagonists MK-801 and AP-5, the non-NMDA receptor antagonist CNQX or the nitric oxide synthase inhibitor L-NAME. In vivo microdialysis analysis showed short-term elevation of extracellular glutamate concentration in the spinal cord after intrathecal injection of TBOA. Furthermore, topical application of TBOA on the dorsal surface of the spinal cord resulted in a significant elevation of extracellular glutamate concentration demonstrated by in vivo glutamate voltametry. The present study indicates that defective spinal glutamate uptake caused by inhibition of glutamate transporters leads to excessive glutamate accumulation in the spinal cord. The latter results in persistent over-activation of synaptic glutamate receptors, producing spontaneous nociceptive behaviors and sensory hypersensitivity. Our results suggest that glutamate uptake through spinal glutamate transporters is critical for maintaining normal sensory transmission under physiologic conditions.

Alteration of glutamatergic (GLU) neurotransmission within the spinal cord contributes to hyperalgesic and allodynic responses following nerve injury. In particular, changes in expression and efficacy of glutamate transporters have been reported. Excitatory, pain transmitting primary afferent neurons utilizing glutamate as an excitatory neurotransmitter project to both superficial (I-II) and deep (III-V) laminae of the dorsal horn. These experiments were designed to examine changes in glutamate uptake occurring concomitantly within the spinal deep dorsal and ventral horn in situ after experimentally induced neuropathic pain. In vivo voltammetry, using microelectrode arrays configured for enzyme-based detection of GLU were employed. Sprague-Dawley rats had either sham surgery or tight ligation of L5 and L6 spinal nerves (SNL). Four to six weeks later, the L4-L6 spinal cord of chloral hydrate-anesthetized animals was exposed, and ceramic-based glutamate microelectrodes equipped with glass micropipettes 50 microm from the recording surfaces were placed stereotaxically at sites within the spinal cord. Pressure ejection of GLU into the ipsilateral L5-L6 spinal cord resulted in a 72% reduction of GLU uptake in SNL rats compared to sham controls in the ipsilateral L5-L6 deep dorsal horn and a 96% reduction in the ventral horn. In contrast, in the same animals, the contralateral L5-L6 or the ipsilateral L4 spinal cord showed no change in glutamate uptake. The data suggest that spinal nerve ligation produced attenuated glutamate uptake activity extending into the deep dorsal and ventral horn. The study suggests that plasticity related to spinal nerve injury produces widespread alteration in glutamate transporter function that may contribute to the pathophysiology of neuropathic pain.

Peripheral nerve injury leads to the activation of spinal cord astrocytes, which contribute to maintaining neuropathic (NP) pain behavior. Fibroblast growth factor-2 (FGF-2), a neurotrophic and gliogenic factor, is upregulated by spinal cord astrocytes in response to ligation of spinal nerves L5 and L6 (spinal nerve ligation [SpNL]). To evaluate the contribution of spinal astroglial FGF-2 to mechanical allodynia following SpNL, neutralizing antibodies to FGF-2 were injected intrathecally. Administration of 18 microg of anti-FGF-2 antibodies attenuated mechanical allodynia at day 21 after SpNL and reduced FGF-2 and glial acidic fibrillary protein mRNA expression and immunoreactivity in the L5 spinal cord segment of rats with SpNL. These results suggest that endogenous astroglial FGF-2 contributes to maintaining NP tactile allodynia associated with reactivity of spinal cord astrocytes and that inhibition of spinal FGF-2 ameliorates NP pain signs.

Neuropathic pain involves co-regulation of many genes and their translational products in both peripheral and central nervous system. We used proteomics approaches to investigate expressional changes in cytosolic protein levels in rat brainstem tissues following ligation of lumbar 5 and 6 (L5, L6) spinal nerves, which generates a model of peripheral neuropathic pain (NP). Proteins from brainstem tissue homogenates of NP and SHAM animals were fractionated by two-dimensional (2-DE) gel electrophoresis to produce a high-resolution map of the brainstem soluble proteins. Proteins showing altered expression levels between NP and SHAM were selected. Isolated proteins were in-gel trypsin-digested and the resulting peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Using the mass spectrometric data, we were able to identify 17 proteins of interest through searches of the Swiss-Prot and NCBi nonredundant protein sequence database. Several of the identified proteins, including fatty acid binding protein-brain (FABP-B), major histocompatibility complex (MHC) class 1, T-cell receptor (TCR) alpha chain, and interleukin-1 (IL-1), showed significantly higher levels in the NP rat brainstem. Proteomic analysis has identified several proteins with differential expression levels in NP as compared to SHAM. However, the function of the proteins identified is postulated; therefore, further experiments are required to determine the true role of each protein in NP.

In peripheral neuropathy (PN), dorsal column (DC) fibers that synapse in the nucleus gracilis (NuGr) mediate expression of mechanical allodynia and have increased expression of brain-derived neurotrophic factor (BDNF). Neurotrophins (NTs) are implicated in pathology or repair in PN. To assess NTs in the NuGr in PN, mRNA expression of BDNF, nerve growth factor (NGF), and NT receptors TrkA, TrkB, and p75 was determined 1 week after ligation of L5 and L6 spinal nerves (SNL). Laser capture microdissection was used to collect NuGr tissue followed by reverse-transcription (RT)-PCR. TrkA, TrkB, and NGF mRNA levels decreased, whereas p75 mRNA increased, in ipsilateral SNL NuGr compared with SHAM; BDNF was undetectable. Decreased Trk mRNA may result in decreased NT activity in the NuGr. The p75 receptor influences Trk activity and cell survival, thus its role in PN warrants further investigation.

Spinal nerve ligation results in dramatic changes in spinal cord primary C-afferent fibers, which include atrophy with an accompanied decrease in calcitonin-gene-related peptide (CGRP). These changes parallel the activation of astrocytes, which have been implicated in the ensuing neuropathic pain states. As part of an effort to elucidate the role of the downstream effectors of astrocyte reactivity in the context of allodynia, the expression of fibroblast growth factor-2 (FGF-2) was examined following tight ligation of L5 and L6 spinal nerves. FGF-2 is a pleiotropic cytokine that is synthesized and secreted by neurons and astrocytes. FGF-2 immunoreactivity was increased in ipsilateral dorsal horn reactive astrocytes at 1 and 3 weeks following nerve ligation. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) of laser-captured dorsal spinal cord sections revealed an increase in FGF-2 mRNA in the dorsal horn ipsilateral to nerve injury compared to contralateral and SHAM. Furthermore, an increase in FGF-2 mRNA in ispilateral dorsal root ganglia (DRG) was seen by in situ hybridization. These results demonstrate that, in response to ligation-induced injury of sensory neurons, FGF-2 is upregulated in both DRG neurons and in spinal cord astrocytes, suggesting neurotrophic functions of this growth factor following peripheral nerve lesion and possibly in astrocyte-related maintenance of pain states.

Drugs that inhibit reuptake of monoamines are frequently used to treat pain syndromes, e.g. neuropathy or fibromyalgia, where mechanical allodynia is present. Several lines of evidence suggest the involvement of supraspinal sites of action of these drugs. However, a direct study of supraspinal serotonin (5-HT) or norepinephrine (NE) release in an animal model in which allodynia is expressed, e.g. neuropathy, has not been done. The ventrobasal (VB) thalamus and the hypothalamus are major supraspinal projection regions for spinal neurons that transmit nociceptive information and are innervated by monoaminergic fibers. This study determined if peripheral neuropathy would induce changes in extracellular monoamines in VB thalamus and hypothalamus. Male Sprague-Dawley rats had spinal nerve roots L5 and L6 tightly ligated (neuropathic rats; NP) or sham (SHAM) surgery; contralateral and ipsilateral VB thalamus and contralateral hypothalamus were dialyzed with modified artificial cerebral spinal fluid (aCSF), with and without fluoxetine. NP rats had significantly decreased 5-HT content in dialysates of the contralateral VB thalamus compared with SHAM rats with (82% decrease) or without (63% decrease) fluoxetine in the perfusion medium over the 180 min of the study. There were no differences in the ipsilateral VB thalamus. In contrast, release of 5-HT was unchanged in the hypothalamic dialysates of SHAM vs. NP rats. NE release was not different in dialysates of either the VB thalamus or hypothalamus of SHAM vs. NP rats. Synthesis of 5-HT, as assessed by accumulation of 5-hydroxytrytophan after treatment with an L-amino acid decarboxylase inhibitor, was not different between NP and SHAM rats in VB thalamic and hypothalamic brain tissue. This study is the first to demonstrate changes in monoamine release supraspinally in NP rats. The differential effect between VB thalamus and hypothalamus suggests that a terminal field change may be involved. Putative mechanisms for mediating this change include alterations of GABA-ergic systems and/or plasticity related to alterations in N-methyl-D-aspartate receptor activation and nitric oxide release related to afferent hyperactivity induced by neuropathic pain., (Copyright 2002 International Association for the Study of Pain)

Methiothepin, a nonselective 5-HT receptor antagonist was utilized to explore the 5-HT modulation of dorsal vagal complex-TRH (thyrotropin releasing hormone) analogue stimulated gastric functional parameters. Intracisternal methiothepin pretreatment (200, 0.1 nmol) produced significant inhibition (70%, 44%, respectively) of the TRH analogue [p-Glu-His-(3,3'-dimethyl)-Pro NH2; RX 77368 (12 pmol)]-induced gastric acid output compared to vehicle pretreatment. Intracisternal pretreatment with methysergide (nonspecific 5-HT receptor antagonist) or combined cyanopindolol (5-HT(1A and 1B) receptor antagonist)+ritanserin (receptor antagonist of the 5-HT(2) family) did not alter the dorsal vagal complex-RX 77368 response. Unilateral dorsal vagal complex pretreatment with methiothepin (50 nmol/50 nl) attenuated ipsilateral dorsal vagal complex-TRH analog (12 pmol) induced gastric secretory response by 57%. The gastric secretagogue response to stimulation of the raphe obscurus (mediated by TRH release into the dorsal vagal complex) was inhibited 50% by pretreatment with intracisternal dorsal medullary methiothepin (0.1 nmol/10 microl). Intracisternal methiothepin (200 nmol/20 microl) also attenuated (a) dorsal vagal complex-glutamate (60 nmol/30 nl) stimulated gastric acid secretion and (b) gastric motility stimulated by dorsal vagal complex-RX 77368 (12 pmol/30 nl). The data suggest that other properties of methiothepin, alone or in addition to its 5-HT receptor antagonist effect, mediate its inhibitory actions at the dorsal vagal complex.

Serotonin (5-HT) interacts with thyrotropin-releasing hormone (TRH) at the dorsal vagal complex (DVC) to augment TRH-induced stimulation of gastric acid secretion. To investigate the 5-HT receptor family involved in the augmentation response, prototypical 5-HT receptor-selective agonists (146 pmol) were coinjected with the TRH analog RX-77368 (RX; 12 pmol) into the rat DVC in a 30-nl volume. The DVC coordinates were 0.2 mm anterior, 0.2 mm right, 0.6 mm ventral with respect to the calamus scriptorius. Coinjection of RX with the 5-HT agonists 5-carboxyamidotryptamine (5-CT) or (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI; 5-HT2 agonist) produced a 183 or 103% increase in gastric acid output compared with the RX injection alone. In contrast, coinjection of 2-methyl-5-HT (5-HT3 agonist) with RX produced no effect on RX-induced increase in gastric acid secretion. Moreover, coinjection of SC-53116 (5-HT4 agonist) decreased the gastric acid output by 45% compared with the RX response itself. Examination of the RX/5-HT agonist coinjection response in more rostral regions of the DVC using the same doses (5-CT/RX or DOI/RX) revealed that only 5-CT was effective in producing the augmented response to TRH analog. The results suggest that activation of 5-CT- or DOI-sensitive receptors augments, and of 5-HT4 receptors inhibits, the gastric acid response to TRH analog injected into the DVC. Thus the integrated response to several serotonin receptor subtypes may mediate changes to the TRH response induced by 5-HT at the DVC.

The pancreatic polypeptide family of hormones and neurotransmitters including pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) are emerging as potent central regulators of gastric function. There is, however, considerable debate concerning the mechanisms and even the direction of effects mediated by these peptides. Good evidence exists showing that PYY is the 'enterogastrone' released by the ileum after feeding which acts on vagal reflex control circuits to reduce gastric motility (i.e. the 'ileal brake'). However, equally convincing evidence is available to suggest that PYY and its close structural relative NPY may act in the same region of the brain to increase gastric motility through vagal mechanisms. We hypothesize that the confounding observations are due to agonist effects on two different receptor types-Y1 and Y2, which are both present in the dorsal vagal complex (DVC) but may be accessed differentially by peripheral humoral (PYY) vs central (NPY) pathways. In our initial approach to this problem, we studied the effects of NPY, PYY, Y1 and Y2 agonists microinjected into the DVC on gastric motility in the stimulated (by central thyrotropin-releasing hormone (TRH) and basal conditions. Our results show that Y2 agonist applied to the DVC during conditions of TRH-stimulated gastric motility mimicked the suppressive effects of PYY applied under the same conditions. Under basal conditions, Y2 agonist has no effect on motility. The DVC effect of the Y1 agonist is the opposite; Y1 agonist has no further effect to stimulate gastric motility in the TRH stimulated condition while Y1 agonist strongly stimulates motility from the basal condition. The effects of NPY depend upon the condition of study. Under TRH stimulation (maximal motility), NPY in the DVC reduces (but does not completely suppress) gastric motility while in the basal state, NPY is a strong activator of motility. These results are discussed in terms of the possible differential localization of Y1 vs Y2 receptors within the DVC and in terms of recent findings suggesting that PYY is rapidly converted to a Y2 agonist by a ubiquitous dipeptidyl aminopeptidase (DAP-IV).

1. The purpose of this study was to examine the change in gastric acid output and gastric erosion formation produced by inducing gastric enterochromaffin-like (ECL) cell hyperplasia in female rats. 2. Rats were treated with vehicle or ranitidine (1,200 mumol/kg/day x 4 wks) administered via SC Alzet minipumps. Experiments were performed 24 hours after removing the minipump, when the inhibitory effect of ranitidine on gastric acid secretion had been lost. 3. Basal gastric acid secretion was 7-fold higher in chronic ranitidine animals than in sham control. 4. Both total and net gastric acid secretions stimulated by carbachol/pentagastrin infusion or histamine injection were significantly higher in the chronic ranitidine animals than in controls. 5. By contrast, intracisternal injection of the chemical vagal stimulant RX77368 (100 ng) resulted in no net increase in acid output of recovered ranitidine-pretreated group. 6. No significant changes in gastric erosions produced experimentally by cold exposure plus restraint or indomethacin pretreatment were noted in recovered chronic ranitidine animals compared to sham controls. 7. These findings suggest that achlorhydria-induced ECL cell hyperplasia augments both basal and stimulated gastric acid secretory function. The histamine results implicate an enhanced parietal cell mass, upregulation of H2 receptors, and/or second-messenger events at the parietal cell as the mechanism for the enhanced gastric secretory response.

Despite many reports that serotonin (5-HT) inhibits gastric acid output, the role and mechanism of action of endogenous 5-HT to modulate gastric secretion remain unclear. Vagal stimulation enhanced the basal rate of 5-HT release into both the gastric lumen (600%) and the portal circulation (265%) of the rat. The peak rate of 5-HT release into the portal circulation was 1,000-fold higher that luminal release (12 micrograms/min and 1.2 ng/min, respectively). To elucidate site(s) of action of 5-HT to inhibit acid secretion, several approaches were taken. Intraluminal perfusion of exogenous 5-HT to encompass enhanced levels seen after vagal stimulation did not reduce gastric acid output. In contrast, administration of systemic 5-HT, which raised portal venous 5-HT to similar levels as vagal stimulation, had a marked antisecretory effect. Chemical or surgical ablation of enteric or sympathetic nerves innervating the stomach did not attenuate the inhibitory effect of exogenous 5-HT on gastric acid output. The antisecretory effect of systemic 5-HT was insensitive to pretreatment with piroxicam, doxantrazole, close gastric intra-arterial sodium nitroprusside, somatostatin monoclonal antibody, or bilateral adrenalectomy. The results suggest that 5-HT is released from endogenous stores into the portal circulation in sufficient quantities after vagal stimulation to alter gastric physiology and that its action is independent of the autonomic nervous system, gastric mucosal prostaglandins or somatostatin, mucosal mast cell or adrenal constituents, or changes in gastric mucosal blood flow.

Convergent evidence suggests that thyrotropin-releasing hormone (TRH) is a principal regulator of several vagally mediated gastric responses. Serotonin (5-HT) interacts with TRH in the dorsal vagal complex (DVC) to augment gastric acid secretory responses. This study investigated the ability of 5-HT to alter other gastric responses mediated by TRH administration into the DVC. Co-injection of 5-HT (7.9 pmol) and the TRH analogue RX-77368 (0.66 pmol) produced a 117% enhancement in 1-h gastric acid output compared with rats treated with RX-77368 (0.66 pmol) alone into the DVC. In contrast, coadministration of RX-77368 (4 pmol) with various doses of 5-HT (0.0048-480 pmol) was ineffective in significantly altering stimulation of gastric antral motility produced by RX-77368 (4 pmol) alone. The effect of a lower dose of DVC RX-77368 (0.66 pmol) on gastric motility was also not changed by 5-HT coadministration. Moreover, the cytoprotective effect of DVC RX-77368 (1.5 pmol) on oral ethanol-induced gastric mucosal lesions was reversed by 5-HT coadministration (54 or 18 pmol). The results suggest that activation of 5-HT receptors in the DVC can augment, not affect, and attenuate DVC TRH analogue-stimulated gastric acid secretion, antral motility, and cytoprotection, respectively.

Peptide YY (PYY) has been shown to effectively regulate gastrointestinal function when administered intravenously. In the present study, the effect of intracisternal (i.c.) injection of rat PYY on the rate of gastric emptying was investigated in conscious animals. Vehicle, bombesin or different doses of PYY was given i.c. after animals had been given an oral liquid meal. A dose-dependent inhibition of gastric emptying was observed following the injection of PYY (400 fmol, 40 fmol and 40 amol; in 10 microliters) into the cisterna magna. The 400 fmol dose of PYY produced an inhibition of similar magnitude as that caused by bombesin (600 fmol in 10 microliters). Furthermore, subdiaphragmatic vagotomy resulted in an elimination of the inhibitory PYY effect on gastric emptying, suggesting that PYY action to inhibit gastric function depends on intact vagal pathways.

Serotonin interacts with TRH at the dorsal vagal complex (DVC) to augment gastric functional parameters. To ascertain physiologic relevance, patterns of stimulated release at the terminal field were characterized. Stimulation of the nucleus raphe obscurus (nRO) by kainic acid (423 pmol/10 nol) produced marked release of serotonin into dorsal medullary dialysates containing the DVC in freely fed, but no 24-h fasted rats. Probe infusion of kynurenic acid (1 mM), but not acute bilateral cervical vagotomy attenuated nRO-stimulated serotonin release in fed animals. The results suggest that the fed state facilitates serotonin release into the dorsal medulla by a mechanism mediated by activation of excitatory amino acid receptors in the dorsal medulla. Enhanced serotonergic neurotransmission at the DVC may comprise a heretofore unrecognized component of the integrated vago-vagal response to a meal.

Serotonin (5-hydroxytryptamine, 5-HT) is a neuroendocrine component of the gastrointestinal tract. 5-HT1A receptors exist both in the brain and have been demonstrated autoradiographically in high density in the rat stomach. However, the physiologic role of 5-HT1A receptors in modulating gastric function is not known. The effect of the selective 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT), on gastric acid secretory function was compared to 5-HT in acute, urethane-anesthetized gastric-fistulated rats during pentagastrin infusion. 5-HT inhibited, but 8-OH-DPAT stimulated, gastric acid secretion in a dose-dependent manner. Bilateral cervical vagotomy or celiac ganglionectomy did not reverse the effect of 8-OH-DPAT on acid secretion. However, the enhancement of acid by 8-OH-DPAT was attenuated by acute adrenalectomy or close intra-arterial administration of spiperone, but not idazoxan. Thus, the data suggest that the selective 5-HT1A receptor agonist 8-OH-DPAT may augment gastric secretory function via an adrenal-dependent mechanism.

Pancreatic polypeptide (PP) has been shown to alter gastrointestinal functions, including increased gastric acid secretion and motility following brain stem injections of PP. The present study investigated the effect of an intracisternal injection of PP on the rate of gastric emptying. Additionally, the efficacy of the rat and bovine forms of the peptide was compared. Rats anesthetized with ether received an intracisternal injection of rat PP, bovine PP, or vehicle and, upon regaining consciousness, were fed a liquid test "meal." Intracisternal rat PP produced a marked enhancement in gastric emptying compared with control animals. Bovine PP, at doses equimolar to or three times greater than the effective rat PP dose, produced no change in gastric emptying. Pretreatment with systemic atropine prior to central injection of rat PP eliminated the stimulation of emptying, suggesting that PP acts through a cholinergic mechanism. When equimolar doses of rat or bovine PP were microinjected directly into the dorsal vagal complex, the region containing PP receptors, both were capable of stimulating antral motility. The response to bovine PP, however, was delayed and reduced compared with that seen following rat PP. The results suggest that rat PP strongly stimulates gastric emptying in rats and that bovine PP, depending on the route of administration, is either ineffectual or a weaker agonist for central PP receptors.

5-Hydroxytryptamine (serotonin, 5-HT) is released from the gastrointestinal tract by vagal stimulation. This biogenic amine produces many alterations in gastric functional parameters, including inhibition of gastric acid secretion. This study was designed to characterize the 5-HT receptor subtype modulating gastric acid secretion. In urethane-anesthetized acute gastric fistula rats, systemic 5-HT (3.5 mumol/kg i.v.) inhibited acid output stimulated by pentagastrin infusion by 58%. Close gastric intra-arterial (i.a.) injection of methysergide, methiothepin, metergoline or spiperone but not tropisetron, renzapride or ritanserin was effective in reversing 5-HT-induced inhibition of acid secretion. Close i.a. administration of the potent 5-HT1A/1B antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine displayed partial agonist properties in this model, but did not antagonize 5-HT-induced inhibition of acid output. In a study of 5-HT agonists given close i.a. to the gastric circulation, 5-HT (0.88 mumol/kg) inhibited acid secretion by 48%. A 3-fold higher dose (2.6 mumol/kg) of the general 5-HT1 agonist, 5-carboxamidotryptamine (5-CT), was needed to inhibit acid secretion significantly. In contrast, neither the selective 5-HT1A agonist (+-)-8-hydroxy-2-(n-dipropylamino)-tetralin nor 5-HT2 agonist (+-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (0.88 or 2.6 mumol/kg) attenuated gastric acid secretion. Thus, the data suggest that the site mediating inhibition of acid secretion by exogenous 5-HT belongs to the 5-HT1 family, but may not be of the 5-HT1A subtype.

Intracisternal (ic) injection of the neutral endopeptidase-24.11 inhibitor phosphoramidon (1-100 nmol) produced a dose-dependent inhibition of gastric acid secretion in 2-h pylorus-ligated rats. The response resulted from a reduction in acid concentration and volume. Likewise, ic injection of another neutral endopeptidase-24.11 inhibitor Zincov (200 nmol) produced a 63% inhibition in gastric acid output. In contrast, neither intravenous injection of phosphoramidon (100 nmol) nor ic injection of the aminopeptidase inhibitor amastatin (100 nmol) produced any change in gastric acid secretion. The inhibitory effect of ic phosphoramidon (10 nmol) was not reversed by a dose of naloxone sufficient to antagonize the acid inhibitory effects of ic [D-Ala2-D-met5]enkephalinamide (8.5 nmol). Moreover, phosphoramidon-induced inhibition of acid was not reduced by the centrally effective bombesin antagonist N-acetyl-GRP(20-26)-O-CH3 or by reserpine pretreatment at a dose effective to antagonize ic neurotensin-induced inhibition in acid secretion. These results suggest that an endogenous neutral endopeptidase-24.11 sensitive substrate may act in the brain to inhibit gastric acid output by mechanisms independent of CNS opiate, bombesin or neurotensin activity.

The effects of serotonin (5HT) and a thyrotropin-releasing hormone (TRH) analogue, RX77368, on vagal control of gastric acid secretion were studied. Microinjection of RX77368 (0.66 pmol in 10 nl), but not 5HT (8 pmol in 10 nl), into the dorsal vagal complex (DVC) evoked a significant increase in acid output. When the same doses of RX77368 and 5HT were co-injected, the amount of acid secreted was significantly greater than that due to RX77368 alone. Thus, 5HT and the TRH analogue interact within the DVC to enhance vagal stimulation of acid secretion. The study suggests a possible functional significance of raphe TRH/serotonergic tracts projecting to the DVC.

Central injection of TRH or its metabolically stable analogue RX 77368 has been demonstrated to produce a vagal-dependent stimulation in gastric acid secretion. Accumulating evidence exists regarding the interaction of serotonin (5HT) with TRH containing neuronal systems. This study was performed to assess the effect of pretreatment with the 5HT uptake inhibitor fluoxetine on the TRH analogue-induced gastric acid secretory response. Systemic fluoxetine (30 mumol/kg, i.v.) produced a 43-85% increase in the intracisternal RX 77368 (78-780 pmol)-induced gastric acid output, while not affecting the basal acid response. The acid response to a lower dose of RX 77368 (26 pmol) was not altered. In addition, intracisternal fluoxetine (180 nmol) produced a 71% augmentation of the acid secretory response of i.c. RX 77368 (260 pmol). Intracisternal injection of lower doses (60, 120 nmol), or intravenous injection of 180 nmol of fluoxetine was ineffective in altering the intracisternal RX 77368-induced acid response. Pretreatment with the noradrenergic or dopaminergic uptake inhibitor desipramine or GBR 12909 did not alter the RX 77368-stimulated gastric acid secretory response. The results show that fluoxetine pretreatment potentiates the effect of intracisternal RX 77368 on acid secretion. The effect appears to be impulse dependent, and central sites of action are involved. The data suggest an interaction of synaptic serotonin with a RX 77368-elicited event (activation of TRH receptors, second messenger systems and/or firing of the motor vagus) results in potentiation of the RX 77368-induced gastric response.

1. Propyl-methylenedioxyindene (pr-MDI) is an intracellular calcium antagonist which inhibits cold-restraint stress ulceration at subcardiovascular doses (less than or equal to 30 mg/kg). It also inhibits gastric acid secretion evoked by RX77368 (a stable analog of thyrotropin-releasing hormone, the putative mediator of cold-restraint stress ulcers). 2. The objective of this investigation was to localize the site of the inhibitory effect of pr-MDI on gastric acid secretion stimulated by intracisternal (i.c.) administration of RX77368 (100 ng) in rats. 3. Peripheral administration of pr-MDI (30 mg/kg i.p. or i.v.) inhibited the elevated basal and the RX 77368-induced acid secretion in conscious 2-hr pylorus-ligated rats. This effect was completely blocked in anesthetized (urethane or pentobarbital) acute gastric fistula rats. 4. Intracisternal administration of pr-MDI (0.1-1 mumol) produced a dose-related inhibition of acid secretion in conscious pylorus-ligated rats. However, urethane anesthesia completely blocked the inhibitory effect of i.c.-administered pr-MDI (1 mumol) on RX77368-induced acid secretion. 5. Since previous studies indicate that anesthesia does not inhibit peripheral actions of pr-MDI (e.g. the antiarrhythmic effect), the convergent evidence suggests that the inhibitory effect of pr-MDI on gastric acid secretion is mediated primarily via the central nervous system.

Intracisternal injection of the thyrotropin releasing hormone (TRH) analogue RX 77368 (100 ng) or the somatostatin analogue ODT8-SS (1 microgram) produced an 82% and 101% increase in gastric acid secretion in 2 h pylorus-ligated rats. In contrast, dissimilar effects were produced by intracisternal injection of these peptides on the secretion of serotonin into the gastric lumen. Intracisternal RX 77368 (100 ng) produced a 496% increase in intraluminal serotonin release, while in contrast, intracisternal ODT8-SS (1 microgram) produced a 78% inhibition in intraluminal serotonin release. Bilateral adrenalectomy reversed the stimulatory effect of intracisternal RX 77368 (100 ng) on serotonin, but not acid release. The data reveal a difference in the ability of the two peptides, which act as gastric secretagogues, to produce intraluminal acid and serotonin release, and suggest that combined activation of the vagus and the adrenal gland are important in mediating basal and RX 77368-stimulated serotonin release into the gastric lumen. In particular, differential effects on adrenal catecholamine release are implicated in the divergent effects of the two peptides.

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

Propyl-methylenedioxyindene (pr-MDI; 30 mg/kg, i.p.), an intracellular calcium antagonist, significantly reduced the number and size of erosions per stomach induced by cold-restraint stress by 69% and 86%, respectively. Our previous findings indicate that the antiulcer activity of pr-MDI is highly correlated with its inhibitory effect on gastric motor activity. Since central TRH is suggested as the brain mediator responsible for cold-restraint stress gastric ulcers in rats, the inhibitory action of pr-MDI was evaluated in the TRH-induced gastric lesion model. Pr-MDI (30 mg/kg) did not reduce the gastric erosions induced by intracisternal administration of 100ng RX77368, a stable thyrotropin-releasing hormone (TRH) analogue, even though it abolished the RX77368-induced stimulation of gastric emptying, gastric acidity, and acid output. Since pr-MDI (30 mg/kg, i.p.) significantly inhibited the stimulation of gastric motility by both cold-restraint stress and TRH, but only cold-restraint stress-induced gastric erosions were effectively reduced by the drug, the present findings suggest a possible dissociation between the ulcerogenic mechanisms of cold-restraint stress and intracisternal administration of TRH.

Since propyl-methylenedioxyindene (pr-MDI) exhibits significant protective effects against stress-induced ulcers in rats at subcardiovascular doses (10-30 mg/kg, i.p.), the aim of the present study was to explore the effect of this intracellular calcium antagonist on cysteamine-induced duodenal ulcers at the same low doses. Duodenal ulcers were induced in rats with a single dose of cysteamine (425 mg/kg, s.c.), which produced an 80% ulcer incidence within 24 h without affecting gastric acid concentration. Administration of pr-MDI (10 and 30 mg/kg, i.p.) at 0, 6 and 12 h post-cysteamine did not afford protection against ulceration. On the other hand, atropine (10 mg/kg, s.c., administered at 0, 6 and 12 h post-cysteamine) resulted in a 69% inhibition of ulceration, and the antacid Maalox (2 ml, administered p.o. at 0, 2, 4, 6 and 12 h post-cysteamine) completely prevented ulceration. The failure of pr-MDI to protect against duodenal ulceration is discussed in relation to its pharmacological mechanism of action and the pathogenetic mechanism of action of cysteamine.

Folic acid (FA) and certain of its reduced congeners produce excitatory effects when applied to neuronal tissue. Recent evidence has suggested that folates have other biological properties in common with the excitatory amino acids. The purpose of this study was to determine the activity of folate compounds in a system sensitive to excitatory amino acids. Bilateral injection of folic acid into the nucleus accumbens resulted in a marked increase in locomotor activity at doses of 2.5 and 5 micrograms. Larger doses resulted in behavioral responses, such as body tremor and labored breathing, which interfered with the locomotor response. Similarly, 5-formyltetrahydrofolic acid (FTHF) produced a marked hypermotility response after bilateral injection into the nucleus accumbens (2.5-25 micrograms), while dihydrofolic acid, tetrahydrofolic acid, and 5-methyltetrahydrofolic acid were ineffective. Pretreatment with reserpine (10 mg/kg, i.p.) markedly reduced the hypermotility response elicited by folic acid and FTHF as did pretreatment with haloperidol in both peripheral (0.8 mg/kg) and direct (5 micrograms) injection into the nucleus accumbens. In addition, injection of muscimol (30 ng), which depresses hypermotility induced by dopamine and amphetamine, produced a significant decrease in the hypermotility response produced by folic acid. In contrast, pretreatment with phentolamine (5 mg/kg, i.p.) or propranolol (4 mg/kg, i.p.) did not decrease folic acid or FTHF-induced responses. These results suggest that folic acid and FTHF produce an increase in locomotor activity by facilitating dopaminergic neurotransmission in the nucleus accumbens, possibly by inducing the release of dopamine from the nerve terminals. Thus, these folates have effects similar to those of the excitatory amino acids when injected into the nucleus accumbens.

There is clear evidence in rats that TRH acts in the brain to stimulate gastric acid, pepsin, and serotonin secretion, mucosal blood flow, contractility, emptying, and ulceration through activation of parasympathetic outflow to the stomach (TABLE 3). A number of TRH analogues, including some devoid of TSH-releasing activity, mimic the effects of TRH. The most sensitive TRH sites of action to elicit gastric acid secretion and motility are located in the dorsal vagal complex and include the dorsal vagal, nucleus tractus solitarius, and nucleus ambiguus. The gastrointestinal tract is one of the most responsive visceral systems to the central effects of TRH, because doses in the range of 1-10 pmol in the dorsal vagal complex stimulate gastric function, whereas stimulation of cardiovascular and respiratory function on microinjection of the brainstem nuclei requires higher doses. Although fewer investigations have been carried out in other species, evidence from the available data clearly indicates that TRH acts in the brain to increase gastric secretion and motility in the rabbit, sheep, and cat. Lack of stimulation of gastric acid secretion after third ventricle injection in the dog may be related to species difference or to rapid degradation of the peptide before it reaches its site of action. TRH acts centrally to stimulate gastric function and also intestinal secretion, motility, and transit as reported mostly in rabbits (TABLE 3). TRH produces enteropooling and release of serotonin in portal blood, increases duodenal and intestinal contractility and colonic transit, and elicits diarrhea. All these effects were shown to be vagally mediated. Stimulation of intestinal motility and transit by central injection of TRH has been observed in rats and sheep. The biological activity of centrally injected TRH is well correlated with the presence of TRH immunoreactivity and receptors in the dorsal vagal complex containing afferent and efferent connections to the stomach. Moreover, endogenous release of brain TRH in rats mimics the stimulatory effect of centrally injected TRH on gastric function. Although the lack of a specific TRH antagonist has hampered assessment of the physiological role of TRH, converging neuropharmacological, neuroanatomical, and physiological findings support the concept that TRH in the dorsal vagal complex may play a physiological role in the vagal regulation of gastrointestinal function.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of intracisternal injection of CRF antagonist, alpha-CRF 9-41, on the inhibition of gastric acid secretion elicited by intracisternal injection of corticotropin-releasing factor (CRF) and stress were investigated in conscious pylorus-ligated rats. Intracisternal injection of the alpha-helical CRF 9-41 (50 micrograms) did not influence basal gastric secretion, but injected concomitantly with intracisternal CRF (5 micrograms), completely blocked CRF (5 micrograms)-induced inhibition of gastric secretory volume, acid concentration and output. Intracisternal injection of alpha-helical CRF 9-41 (3, 10, 50 micrograms) produced a dose-related reversal (0, 52 and 100%) of brain surgery-induced inhibition of gastric acid output. By contrast intravenous injection of CRF antagonist (50 micrograms) did not inhibit gastric hyposecretory response to brain surgery. These data suggest that endogenous CRF in the brain may mediate stress-induced gastric hyposecretion in the rat.

Intracisternal injection of thyrotropin-releasing hormone (TRH)-Gly (pGlu-His-Pro-Gly) produced a dose-dependent (1-100 micrograms) stimulation of gastric acid secretion in urethane-anesthetized rats implanted acutely with a gastric fistula. The peak response occurred 20-30 min after intracisternal injection and lasted for more than 2 h. Intravenous injection of TRH-Gly (100 micrograms) did not modify gastric acid secretion. Following intracisternal injection of TRH-Gly, a peak elevation of both TRH-Gly and TRH levels is observed in the cerebrospinal fluid (CSF) within 15 min. Thereafter, TRH values are returned to basal levels at 75 min after the injection, whereas TRH-Gly concentrations remain significantly elevated throughout the 2-h period of measurement. Compartmental analysis revealed that CSF conversion of TRH-Gly to TRH was only 0.0072%/min. Medullary coronal sections containing the dorsal vagal complex and the raphé nucleus revealed increased content of TRH-Gly, but not TRH, 40 min after administration of TRH-Gly at an intracisternal dose effective in stimulating gastric acid secretion (100 micrograms). In addition, TRH but not TRH-Gly (10(-7)-10(-5) M) displaced [3H]MeTRH binding from rat medullary blocks containing the dorsal vagal complex. These data suggest that the intracisternal TRH-Gly-induced stimulation of gastric acid secretion is not related to its conversion to TRH in the CSF, or direct activation of TRH receptors in the medulla. The acid secretory response of TRH-Gly may be due to the formation of TRH at the active brain sites, or alternatively to activation of its own specific receptors.

Folic acid (FA) and 5-formyltetrahydrofolic acid (FTHF) have been shown previously to produce a marked stimulation of locomotor activity after bilateral injection into the rat nucleus accumbens. This study was designed to determine whether the hypermotility response produced by the folates is mediated through the activation of excitatory amino acid receptors in the nucleus accumbens. Although FA stimulated locomotor activity, pteroic acid, a congener of FA that lacks the glutamate moiety, was ineffective, suggesting that the glutamate portion of the molecule is essential for the hypermotility response. The N-methyl-D-aspartic acid (NMDA) receptor antagonists, D-alpha-aminoadipic acid, DL-alpha-epsilon-diaminopimelic acid and MgCl2, at doses that attenuated NMDA-induced hypermotility, were ineffective in decreasing the folate-induced hypermotility response. This behavioral observation is consistent with the biochemical observation that the folates, at a 1 mM concentration, were unable to stimulate the release of [3H]acetylcholine from striatal slices, a model system that is sensitive to the activation of NMDA receptors. In contrast to the ineffectiveness of the NMDA antagonists in inhibiting the response to the folates, the antagonist, glutamic acid diethylester, which inhibited the response to quisqualic acid, but not NMDA, also inhibited the response to both FA and FTHF. Two recently characterized dipeptides, gamma-D-glutamylaminomethylsulfonic acid and gamma-D-glutamyltaurine, antagonized the stimulation of locomotor activity produced by quisqualic acid, FA and FTHF. However, these dipeptides also inhibited the response to NMDA, suggesting that these compounds are not able to distinguish between quisqualate and NMDA receptors in the nucleus accumbens.(ABSTRACT TRUNCATED AT 250 WORDS)