Renoprotection by pituitary adenylate cyclase-activating polypeptide in multiple myeloma and other kidney diseases.

Renal involvement in patients with multiple myeloma complicates their treatment and shortens their life-span. The main renal lesion is a tubulointerstitial transformation with fibrosis, frequently associated with cast formation in the distal nephron that results from co-precipitation of pathological immunoglobulin light chains with Tamm-Horsfall proteins. The human renal proximal tubular reabsorption of excessive light chains by endocytosis causes cellular protein overload and activates the transcription factor nuclear factor kappa B (NFkappaB). The activation of NFkappaB promotes the synthesis of inflammatory cytokines and activates signaling pathways, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2, Jun kinase, and p38 MAPK, thus promoting interstitial inflammation and fibrosis. We tested the concept that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/vasoactive intestinal peptide family, could prevent the development of cast nephropathies. PACAP38 inhibited myeloma light chain-induced proinflammatory cytokine expression with greater potency than dexamethasone, and attenuated the resulting cell damage in the renal proximal tubule epithelial cells. The results indicated that its effects are mediated through inhibition of phosphorylation of p38 MAPK and nuclear translocation of the p50 subunit of NFkappaB via both the PAC(1) and VPAC(1) receptors. PACAP was also shown to be efficacious in other common in vivo animal models for kidney hypertrophies, including streptozotocin-induced diabetic nephropathy and gentamicin-induced nephrotoxicity. Thus, our studies suggest that PACAP38 could be used as a cytoprotective agent that would be effective in the treatment of renal tubule injury in multiple myeloma and other chronic kidney diseases.

Pituitary adenylate cyclase-activating polypeptide-induced differentiation of embryonic neural stem cells into astrocytes is mediated via the beta isoform of protein kinase C.

We have found previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases the number of astrocytes generated from cultured mouse neural stem cells (NSCs) via a mechanism that is independent of the cyclic AMP/protein kinase A pathway (Ohno et al., 2005). In the present study, the signaling pathway involved in the differentiation process was further investigated. PACAP-induced differentiation was inhibited by the phospholipase C inhibitor, U73122, the protein kinase C (PKC) inhibitor, chelerythrine, and the intracellular calcium chelator, BAPTA-AM, and was mimicked by phorbol 12-myristate 13-acetate (PMA), but not by 4alpha-PMA. These results suggest that the PACAP-generated signal was mediated via the PACAP receptor, PAC1 stimulated heterotrimeric G-protein, resulting in activation of phospholipase C, followed by calcium- and phospholipid-dependent protein kinase C (cPKC). To elucidate the involvement of the different isoforms of cPKC, their gene and protein expression were examined. Embryonic NSCs expressed alpha and betaII PKC, but lacked PKCgamma. When NSCs were exposed to 2 nM PACAP, protein expression levels of the betaII isoform transiently increased two-fold before differentiation, returning to basal levels by Day 4, whereas the level of PKCalpha increased linearly up to Day 6. Overexpression of PKCbetaII with adenovirus vector synergistically enhanced differentiation in the presence of 1 nM PACAP, whereas expression of the dominant-negative mutant of PKCbetaII proved inhibitory. These results indicate that the beta isoform of PKC plays a crucial role in the PACAP-induced differentiation of mouse embryonic NSCs into astrocytes.

Pituitary adenylate cyclase-activating polypeptide is a potent inhibitor of the growth of light chain-secreting human multiple myeloma cells.

Multiple myeloma represents a malignant proliferation of plasma cells in the bone marrow, which often overproduces immunoglobulin light chains. We have shown previously that pituitary adenylate cyclase-activating polypeptide (PACAP) markedly suppresses the release of proinflammatory cytokines from light chain-stimulated human renal proximal tubule epithelial cells and prevents the resulting tubule cell injury. In this study, we have shown that PACAP suppresses the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. The addition of PACAP suppressed light chain-producing myeloma cell-stimulated interleukin 6 (IL-6) secretion by the bone marrow stromal cells (BMSCs). A specific antagonist to either the human PACAP-specific receptor or the vasoactive intestinal peptide receptor attenuated the suppressive effect of PACAP on IL-6 production in the adhesion of human multiple myeloma cells to BMSCs. The secretion of IL-6 by BMSCs was completely inhibited by 10(-9) mol/L PACAP, which also attenuated the phosphorylation of both p42/44 and p38 mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappaB (NF-kappaB) activation in response to the adhesion of multiple myeloma cells to BMSCs, whereas the inhibition of p42/44 MAPK signaling attenuated PACAP action. The signaling cascades involved in the inhibitory effect of PACAP on IL-6-mediated paracrine stimulation of light chain-secreting myeloma cell growth was mediated through the suppression of p38 MAPK as well as modulation of activation of transcription factor NF-kappaB. These findings suggest that PACAP may be a new antitumor agent that directly suppresses light chain-secreting myeloma cell growth and indirectly affects tumor cell growth by modifying the bone marrow milieu of the multiple myeloma.

Treatment of renal failure associated with multiple myeloma and other diseases by PACAP-38.

Myeloma kidney injury is caused by the large amount of light chain (LC) of immunoglobulins produced by cancerous plasma cells through stimulation of proinflamatory cytokines like TNF-alpha and IL-6. PACAP-38 suppressed LC-stimulated cytokine production by tubular epithelial cells in vitro and in vivo, and prevented injury of these epithelial cells. The suppressive effect is comparable or greater than dexamethasone (dex). Although dex produces adverse side effects when it is given for a long time period, PACAP-38 is a natural and safe neuropeptide and no adverse effect has been reported when administered to produce significant biological effects. Furthermore, PACAP-38 suppressed growth of myeloma cells in culture and also suppressed production of their growth factor, IL-6, production from the bone marrow stromal cells that was stimulated by adhesion of myeloma cells. These findings render PACAP-38 worth evaluation as a safe and potent renoprotectant in myeloma kidney as well as a new antitumor agent for myeloma cells.

Presence of PACAP and VIP in embryonic chicken brain.

The aim of the present article was to investigate the occurrence and temporary changes of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and vasoactive intestinal peptide (VIP) in various brain areas of chicken embryos by means of radioimmunoassay. The highest concentrations of PACAP-38 were measured in the brain stem followed by the hypothalamus, cerebellum, and telencephalon. PACAP-38 levels were significantly higher than those of VIP in all examined brain areas. The levels of both PACAP-38 and VIP showed a tendency to decrease until hatching during embryonic development of the chicken.

Short-term fasting differentially alters PACAP and VIP levels in the brains of rat and chicken.

The present article investigated the levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) in the brains of rats and chickens 12, 36, and 84 h after starvation. PACAP levels increased in both species, 12 h after food deprivation in rats, and with a 24-h delay in chickens. VIP levels showed a more complex pattern: a gradual increase in the hypothalamus and telencephalon, and a significant decrease in the brain stem of rats. In chickens, a decrease was observed in every brain area after 36 h of starvation. These data show that PACAP and VIP are differentially regulated and are involved in the regulatory processes under a food-restricted regimen, and are differentially altered in nocturnal and diurnal species.

Comparative anatomy of PACAP-immunoreactive structures in the ventral nerve cord ganglia of lumbricid oligochaetes.

By means of a whole mount immunocytochemical approach, the distribution patterns of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 were identified in the ventral nerve cord (VNC) ganglia of the earthworms Eisenia fetida and Lumbricus terrestris. Each PACAP form appears to occur in a distinct neuron population. Positions of these populations, as well as numbers and sizes of the constituting neurons do not essentially differ between the two species. The data suggest that in Lumbricid Oligochaetes, PACAP-27 and PACAP-38 neuron populations may mediate distinct physiological processes.

Changes in PACAP levels in the central nervous system after ovariectomy and castration.

The aim of the present article was to investigate the influence of gonadectomy on pituitary adenylate cyclase-activating polypeptide (PACAP) levels in different brain areas. In males, there seems to be an inverse relationship between gonadotropins and PACAP in the brain in the acute phase of castration: PACAP levels decreased in almost all brain areas examined within the first week after castration. In females, such pattern was observed in the hypothalamus, brain stem, and temporal cortex. In the pituitary, levels decreased only on the first day after ovariectomy, and later, as in the thalamus, increases were observed. Although the pattern of change showed gender differences, our results provide further evidence that levels of gonadotropins and possibly gonadotropin-releasing hormone influence PACAP levels and that PACAP is involved in the regulation of gonadal functions.

Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP(+/-) and PACAP(-/-) mice than in PACAP(+/+) animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP(+/-) and PACAP(-/-) mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP(+/-) mice was less than that in PACAP(+/+) animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP(+/-) and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP(+/-) mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.

Signaling involved in pituitary adenylate cyclase-activating polypeptide-stimulated ADNP expression.

Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for VIP and has neuroprotective potential. When the VIP paralog, PACAP38 was added to mouse neuron-glia co-cultures, it induced ADNP mRNA expression in a bimodal fashion at subpico- and nanomolar concentrations with greater response at subpicomolar level. The response was attenuated by a PAC1-R antagonist at both concentrations and by a VPAC1-R antagonist at nanomolar concentration only. An IP3/PLC inhibitor attenuated the response at both concentrations of PACAP38, but a MAPK inhibitor had no effect. A PKA inhibitor suppressed the response at nanomolar concentration only. These findings suggest that ADNP expression is mediated through multiple receptors and signaling pathways that are regulated by different concentrations of PACAP.

Potential protective action of pituitary adenylate cyclase-activating polypeptide (PACAP38) on in vitro and in vivo models of myeloma kidney injury.

The most common type of renal injury in multiple myeloma is chronic tubulointerstitial nephropathy associated with casts in tubule lumens, an entity referred to as "myeloma kidney" that often progresses to end-stage kidney diseases. Myeloma kidney is associated with a significant increase in all-cause mortality, yet no effective intervention, except a limited use of steroid, is available. Here, we report that pituitary adenylate cyclase-activating polypeptide with 38 residues (PACAP38) dramatically prevents injury of cultured renal proximal tubule cells caused by myeloma light chains through suppression of proinflammatory cytokines production, by inhibiting p38 MAPK and translocation of NFkappaB via both PAC(1) and VPAC(1) receptors. The suppressive effects of PACAP was as effective as dexamethasone in all of their cytokine assays and demonstrated both in vitro and in vivo. Furthermore, PACAP38 inhibits myeloma cell growth directly and may also indirectly by suppressing production of the growth factor, IL-6, from bone marrow stromal cells, that is stimulated by adhesion of myeloma cells. These findings render PACAP38 worth evaluation as a promising candidate for an effective and safe renoprotectant in myeloma kidney, and possibly other nephropathy, and also as a new antitumor agent in multiple myeloma.

Signaling cascades involved in neuroprotection by subpicomolar pituitary adenylate cyclase-activating polypeptide 38.

In neuronal/glial cocultures, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevented neuronal death induced by gp120, lipopolysaccharide (LPS), or other toxic agents, but the dose response of the neuroprotective effect is bimodal, with a peak at a subpicomolar concentration and another peak at a subnanomolar to nanomolar concentration. Although the signaling cascade involved in neuroprotection by nanomolar concentration of the peptide has been shown to be mediated by activation of cAMP-dependent protein kinase and subsequent activation of mitogen-activated protein kinase (MAPK), the mechanism for neuroprotection by a subpicomolar level of PACAP38 remains elusive. In the present study, the signaling involved in neuroprotection by subpicomolar PACAP38 was studied in rat neuronal/glial cocultures. Addition of PACAP38 stimulated expression and activation of extracellular signal-related kinase-type MAPK with a peak response at 10-13 M; greater concentrations of the peptide induced lesser response. cAMP production also increased at subpicomolar levels of PACAP38, but the level remained unchanged at a level four to five times higher than the base level at concentrations below 10-11 M. cAMP then started increasing again dose-dependently in a range >10-11 M PACAP38. Lipopolysaccharide (LPS)-induced neuronal death, indicated by increased release of neuron-specific enolase, was suppressed by PACAP38 in a bimodal fashion. Neuroprotection by 10-12 M PACAP38 was completely abolished by a MAPK kinase-1 inhibitor, PD98059, and also partially suppressed by Rp-cAMP, a cAMP-dependent protein kinase inhibitor. Moreover, neuroprotection by a nanomolar level of PACAP38 was completely suppressed by Rp-cAMP but not affected by PD98059. We conclude that neuroprotection by subpicomolar PACAP38 is mainly mediated by the signaling pathway involving MAPK activation and partially regulated by cAMP-dependent protein kinase activation. Furthermore, PACAP38 stimulated expression of activity- dependent neuroprotective protein (ADNP), with a peak at 10-13 M. Greater doses of the peptide induced lesser response. However, 10-13 M PACAP38-stimulated expression of ADNP was not affected by PD98059. This suggests that neuroprotection by subpicomolar PACAP38 might be mediated partially by expression of ADNP, but the major events for neuroprotection by subpicomolar PACAP38 remain to be identified.

Pituitary adenylate cyclase activating polypeptide-mediated intracrine signaling in the testicular germ cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is found not only in the brain, but is also abundantly expressed in the testicular germ cells. However, the physiological role of testicular PACAP remains unknown. Autoradiographic studies showed a considerable number of PACAP-specific binding sites in the seminiferous tubules. Immunohistochemistry demonstrated PAC1-receptor (R)-like immunoreactivity (li) in the cytoplasm of round spermatids, aggregated in the acrosome and coexpressed with PACAP-li. Spermatid-enriched fractions were examined for the subcellular localization of PACAP binding sites and PAC1-R-li. The highest levels of PACAP binding sites and PAC1-R-li were found in the cytosolic, followed by the nuclear, and the lowest levels in the membrane fraction. The testicular cytosolic PAC1-R-like protein showed a specific competitive inhibition in the radio-receptor assay for PACAP38 and 27, with a Ki of 0.069 nM and 0.179 nM, respectively. The addition of PACAP to the cytosol of spermatids only slightly activated adenylate cyclase, while it markedly stimulated the expression and activation of ERK-type mitogen-activated protein kinase (MAPK). In the PAC1-R-like protein-depleted cytosol, a PAC1-R-specific agonist, maxadilan, did not activate MAPK, but PACAP and VIP still did. Because VPAC2-R, which binds both PACAP and VIP, is expressed in the testis, the findings suggest that cytosolic VPAC2-R-like proteins are also present and coupled to MAPK. The MAPK activation does not seem to require a heterotrimeric G-protein. Because PACAP and its receptors are coexpressed in the cytoplasm of spermatids, endogenous PACAP may directly interact with the cytosolic PAC1-R-like protein without the ligand being released into the extracellular space. This possibility is supported by the observation that cytosolic endogenous PACAP in spermatids was co-immunoprecipitated with the cytosolic PAC1-R. This mechanism may be called "intracrine," and its physiological significance is discussed.

Deficits in reproduction and pro-gonadotropin-releasing hormone processing in male Cpefat mice.

Cpe(fat/fat) mice are obese, diabetic, and infertile. These animals have a point mutation in carboxypeptidase E (CPE), an exopeptidase that removes C-terminal basic amino acids from peptide intermediates. The mutation renders the enzyme unstable, and it is rapidly degraded. Although the infertility of Cpe(fat/fat) mice has not been systematically investigated, it is thought to be due to a deficit in GnRH processing. We have evaluated this hypothesis and found hypothalamic GnRH levels to be reduced by 65-78% and concentrations of pro-GnRH and C-terminal-extended intermediates to be high. Basal serum gonadotropin contents are similar among wild-type, heterozygous, and homozygous mice. Testis morphology and function are abnormal in older obese Cpe(fat/fat) mice. Matings between homozygous mutants yield a 5% pregnancy rate. By comparison, when 50-d-old Cpe(fat/fat) males are paired with heterozygous females, rates increase to 43%, and they rapidly decrease to negligible levels by 120 d. As fertility declines without accompanying changes in the hypothalamic-pituitary-gonadal axis and before obesity is evident, reproduction is more complex than originally thought. This suspicion is confirmed in 90-d-old Cpe(fat/fat) males, who readily interact with females, but rarely mount and fail to show intromission or ejaculation behaviors. Together, these findings show that CPE is a key enzyme for pro-GnRH processing in vivo; however, the reproductive deficits in Cpe(fat/fat) males appear to be due primarily to abnormal sexual behavior.

The effect of PACAP on rhythmic melatonin release of avian pineals.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a ubiquitous member of the VIP/secretin/glucagon bioactive peptide family. The distribution, concentration of PACAP, and its role in the control of rhythmic melatonin (MT) secretion from chicken pineal gland were studied. In the chicken pineal gland (ChPG), 40ng PACAP/g tissue was measured with radioimmunoassay. No midday-midnight differences in the PACAP content of the ChPG could be detected. Immunohistochemical studies of ChPG showed PACAP immunoreactive nerve fibers in the perivascular space and around the pinealocytes. Neither PACAP-labeled perikarya nor PACAP immunopositivity in the pinealocytes could be detected. In five day perifusion experiments, carried out under darkness, both MT and cAMP showed circadian rhythmic release pattern from explanted ChPG. One hour exposure of ChPG to PACAP induced transient (3-4h) elevation of MT and cAMP release. The responses were dose-dependent in the range from 1 to 100nM PACAP concentrations. The magnitude of the response was independent on the phase of the daily cycle in which PACAP was applied. cAMP levels during normal daily cycle and also PACAP-induced cAMP responses always preceded similar changes of MT by about an hour suggesting that cAMP is an intracellular intermediate in controlling MT release from the ChPG. At the same time PACAP, similarly to VIP, did not shift the phase of the in vitro circadian MT rhythm of the pineals. Our data reveal, that PACAP is present in nerve fibers in the chicken pineals and pineal cells contain functioning PACAP-sensitive receptors. PACAP apparently exerts a modulatory effect of the rhythmic MT release from the chicken pineal but does not modify the intrinsic biological clock in the avian pineal gland. Thus, cAMP-mediated intracellular mechanisms in ChPG are not components of the pineal circadian clock, but intermediaries between the clock-mechanism and MT release and may also be components of clock-independent MT release modifiers.

Rapidly activated microglial cells in the preoptic area may play a role in the generation of hyperthermia following occlusion of the middle cerebral artery in the rat.

Postischemic hyperthermia occurs after the occlusion of the middle cerebral artery (MCAO) with an intraluminal filament in rats. The cause of hyperthermia is presumed to be damage to the preoptic area, which is one of the temperature-regulatory centers of the hypothalamus. In the present study, reactions of microglial cells and astrocytes in the preoptic area were examined during the first 6 h following transient MCAO. Microglial cells and astrocytes were visualized with immunohistochemistry using antibodies against the CR3 complement receptor and the glial fibrillary acidic protein, respectively. One hour after the occlusion, activated microglial cells were observed in both the medial and lateral preoptic areas ipsilaterally, and in the medial preoptic area contralateral to the infarct. Following reperfusion, the activation of microglial cells decreased in the medial preoptic area of both hemispheres, and in the lateral preoptic area there was a loss of immunoreactive microglial cells. Fragmentation of astrocytic processes was detected in the lateral preoptic area, while in the ipsilateral medial preoptic area a moderate swelling was observed. Immunohistochemistry with an antibody against interleukin-1beta (IL-1beta) revealed scattered immunoreactive cells in both the ipsilateral and the contralateral medial preoptic area 2 h after the MCAO. Our results show that microglial activation in the preoptic area coincides with postischemic hyperthermia. However, an exclusive role for IL-1beta in the generation of hyperthermia is unlikely, and other factors are probably also responsible for postischemic hyperthermia.

The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels.

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNAin the three levels of the hypothalamo-hypophyseal-ovarian axis was previously demonstrated using immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the hypothalamus, PACAP is present in neuroendocrine effector cells and in the median eminence. In the anterior pituitary and ovary, PACAP is transiently present during the proestrous stage of the estrous cycle. In the pituitary, PACAP was observed in gonadotropes. In the ovary, PACAP was demonstrated in the granulosa cells of the preovulatory ovarian follicles. The effect of PACAP on luteinizing hormone (LH) secretion was demonstrated in in vivo and in vitro models. In our work we have studied the role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. At the hypothalamic level, PACAP, administered intracerebroventricularly to female rats before the critical period of the proestrus stage, can inhibit LH release and ovulation. Its inhibiting effect is mediated through corticotropin-releasing factor (CRF) and endogenous opioids. PACAP administered to neonatal female rats delayed the onset of puberty by influencing the luteinizing hormone-releasing hormone (LHRH) neuronal system. In the pituitary gland, the release of PACAP depended on the stage of the estrous cycle and on the time of day the animals were sacrificed. On the day of proestrus, the number of PACAP-releasing cells showed a diurnal change with two peaks (in the morning and in the evening). The peak was much higher in the evening at the end of the LH surge than in the morning.

Neuropeptides of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide/growth hormone-releasing hormone/secretin family in testis.

Mammalian testicular development and the maintenance of spermatogenesis are hormone-dependent processes that are controlled by the pituitary gonadotropins and testosterone. Recent studies have demonstrated the presence of many neuropeptides and their receptors in the testis, suggesting that these peptides operate as local regulators of testicular germ cell development and function. Among these testicular neuropeptides, the peptides that belong to the pituitary adenylate cyclase-activating polypeptide (PACAP) family, particularly growth hormone-releasing hormone and secretin, appear to show some unique common features in terms of intratesticular localization and the time of expression during the spermatogenic cycle. However, their precise physiologic roles and mechanisms of action remain unknown. This review analyzes the available information on the functional interactions among the testicular cells that appear to be mediated by locally produced neuropeptides, with a special emphasis on the peptides of the PACAP family.

Adrenomedullin stimulates somatostatin and thus inhibits histamine and acid secretion in the fundus of the stomach.

Adrenomedullin has recently been localized to enterochromaffin-like (ECL) and chief cells in the gastric fundus. It has been proposed that adrenomedullin may play a role in gastric mucosal defense and repair. In the present study, we have used the isolated, luminally perfused mouse stomach and superfused rat fundic segments to examine the effect of adrenomedullin on exocrine and endocrine secretion in this region of the stomach. Addition of adrenomedullin (1 pM to 1 microM) to the isolated mouse stomach caused a concentration-dependent decrease in acid secretion. The EC(50) value was 1.4 x 10(-9) and maximal inhibition of acid secretion was obtained at a concentration of 1 microM (31+/-4% below basal level, P<0.001). In rat fundic segments, superfusion with adrenomedullin (0.1 pM to 0.1 microM) caused a concentration-dependent increase in somatostatin secretion (EC(50), 1 x 10(-10)) that was accompanied by a reciprocal decrease in histamine secretion (EC(50), 1.2 x 10(-11)). Maximal stimulation of somatostatin secretion (60+/-5% above basal level, P<0.001) and inhibition of histamine secretion (50+/-5% below basal level, P<0.01) was obtained at a concentration of 0.1 microM. Changes in acid and histamine secretion induced by adrenomedullin reflected changes in somatostatin secretion and could be abolished by addition of somatostatin antibody. The axonal blocker, tetrodotoxin, also abolished the somatostatin and, consequently, the acid and histamine responses to adrenomedullin, implying that the effect of adrenomedullin on somatostatin secretion was mediated via activation of intramural neurons. We conclude that adrenomedullin, acting via intramural fundic neurons, stimulates somatostatin and thus inhibits histamine and acid secretion. This represents one mechanism by which adrenomedullin might enhance mucosal defense and repair.