Endotypes of pollen-food syndrome in children with seasonal allergic rhinoconjunctivitis: a molecular classification.

BACKGROUND: Pollen-food syndrome (PFS) is heterogeneous with regard to triggers, severity, natural history, comorbidities, and response to treatment. Our study aimed to classify different endotypes of PFS based on IgE sensitization to panallergens. METHODS: We examined 1271 Italian children (age 4-18 years) with seasonal allergic rhinoconjunctivitis (SAR). Foods triggering PFS were acquired by questionnaire. Skin prick tests were performed with commercial pollen extracts. IgE to panallergens Phl p 12 (profilin), Bet v 1 (PR-10), and Pru p 3 (nsLTP) were tested by ImmunoCAP FEIA. An unsupervised hierarchical agglomerative clustering method was applied within PFS population. RESULTS: PFS was observed in 300/1271 children (24%). Cluster analysis identified five PFS endotypes linked to panallergen IgE sensitization: (i) cosensitization to ≥2 panallergens ('multi-panallergen PFS'); (ii-iv) sensitization to either profilin, or nsLTP, or PR-10 ('mono-panallergen PFS'); (v) no sensitization to panallergens ('no-panallergen PFS'). These endotypes showed peculiar characteristics: (i) 'multi-panallergen PFS': severe disease with frequent allergic comorbidities and multiple offending foods; (ii) 'profilin PFS': oral allergy syndrome (OAS) triggered by Cucurbitaceae; (iii) 'LTP PFS': living in Southern Italy, OAS triggered by hazelnut and peanut; (iv) 'PR-10 PFS': OAS triggered by Rosaceae; and (v) 'no-panallergen PFS': mild disease and OAS triggered by kiwifruit. CONCLUSIONS: In a Mediterranean country characterized by multiple pollen exposures, PFS is a complex and frequent complication of childhood SAR, with five distinct endotypes marked by peculiar profiles of IgE sensitization to panallergens. Prospective studies in cohorts of patients with PFS are now required to test whether this novel classification may be useful for diagnostic and therapeutic purposes in the clinical practice.

Evaluation of food-pollen cross-reactivity by nose-mouth cross-challenge in pollinosis with oral allergy syndrome.

BACKGROUND: Oral allergy syndrome (OAS) is often associated with pollen-induced rhinitis, and there are preferential associations between causative substances. If OAS and rhinitis are both immunoglobulin (Ig)E-mediated and there are cross-reacting proteins, it is expected that similar reactions can be elicited in the nose and mouth. In order to test this hypothesis we performed a series of 'cross-challenges' with foods and pollens in both the nose and the mouth. METHODS: Nine patients with ascertained OAS due to vegetables and rhinitis due to pollens were studied. On the first day a nasal challenge with pollen extracts and an oral challenge with fresh food was carried out. After a week, washout nasal challenge with food and an oral challenge with pollens were performed. Immediate symptoms, mucosal tryptase and soluble eosinophil cationic protein (ECP) were assessed after each challenge. RESULTS: The administration of pollen into the nose and food into the mouth elicited symptoms as expected, but the cross-challenge had no clinical effect. In parallel, tryptase and ECP increased after nasal challenge with pollens, whereas foods did not elicit a measurable response. CONCLUSION: The cross-reactivity between foods and pollens, when evaluated at the shock organ, was not clinically evident. This data can be explained with a low concentration of cross-reagent epitopes in pollen extracts and food homogenized because of degradation. The different behaviour upon challenge suggests that different immunological mechanisms may act in the nose and mouth.

Assessment of residual immunoreactivity in red or white wines clarified with pea or lupin extracts.

Vegetable proteins could be a suitable alternative to animal proteins in the clarification of wine, but their residues could represent a risk for subjects with food allergy or intolerance. The aim of this study was to investigate the presence of specific immunoreactivity in red and white wines treated, as must or wine, with vegetable proteins in the clarification process. The proteins considered were prepared from lupins and peas, which are not included among the allergens listed in annex Illbis of Directive 2003/89/EC. The presence of residual immunoreactivity to specific rabbit anti-lupin and anti-pea polyclonal antibodies in treated wines was assessed by electrophoresis (SDS-PAGE) and immunoblotting. Residual protein was not detectable in red wines clarified with lupin, pea or a mixture of pea and lupin proteins or in white wines clarified with pea proteins. A small number of musts treated with lupin or pea proteins and white wines treated with lupin proteins yielded equivocal results, probably because of the presence of interfering material (e.g., sugar-rich proteins from grape and yeast). The use of bentonite as a secondary clarifying agent is therefore recommended since its combination with vegetable proteins is particularly effective in removing overall protein immunoreactivity.

The phospholipid drug glyceryl-phosphoryl-O-serine modulates pituitary adrenocorticotropin and hypothalamic corticotropin releasing hormone in vitro secretion in the aging rat.

We have investigated the effects of the novel phospholipid drug glyceryl-phosphoryl-O-serine (GPS) on pituitary ACTH and hypothalamic corticotropin releasing hormone secretion in vitro in cultures from both 2- and 24 month-old Sprague-Dawley rats. Basal levels of ACTH in primary cultures of pituitary cells from 24 month-old rats were lower than (100 +/- 12 pg/10(5) cells) in 2 month-old rats (207 +/- 18 pg/10(5) cells). Basal medium corticotropin releasing hormone levels in hypothalamic cultures were higher in 24 month-old rats (45 +/- 7 pg/well/20 min.), than in 2 month-old rats (29 +/- 5.5 pg/well/20 min). Treatment of both pituitary cells with corticotropin releasing hormone and hypothalami with serotonin resulted respectively in a significant, concentration-dependent, increase of medium ACTH and corticotropin releasing hormone. However, concentration-response curves for ACTH and corticotropin releasing hormone were shifted to the right in cultures from 24 month-old rats. Incubation with graded concentrations of GPS produced significant increase in medium ACTH and corticotropin releasing hormone in cultures from 24 month-old rats, whereas the drug was ineffective in stimulating secretion of both hormones from 2 month-old rat cells. In addition, the adenylate cyclase stimulator forskolin and the protein kinase C activator oleyl-acyl-glycerol stimulated ACTH secretion in pituicytes from rats of both ages. However, response to oleyl-acyl-glycerol was blunted in pituicytes from 24 month-old rats. Combination of either forskolin or oleyl-acyl-glycerol with GPS resulted in a potentiation of the effect. Our data confirm an impairment of both pituitary ACTH and hypothalamic corticotropin releasing hormone secretion in the aging rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pidotimod on the immune and the neuroendocrine system in the aging rat.

The effects of pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl)carbonyl]-thiazolidine-4-carboxylic acid, PGT/1A, CAS 121808-62-6), a synthetic thymic dipeptide, on immune response in 2 and 24 month-old rats were investigated. Peripheral blood lymphocytes and splenocytes of aging rats treated for 1 week with different doses of pidotimod showed increased rates of mitogen-stimulated proliferation. Also, interleukin-2 production by 24 month-old rat lymphocytes was significantly increased after treatment with the drug. In addition, the response of the hypothalamic-pituitary-adrenal (HPA) axis to interleukin-1 in 2 and 24 month-old Sprague-Dawley rats previously treated with pidotimod was studies. Blood samples were withdrawn--30, 0, 30, 60, 90 and 120 min after interleukin-1 injection. Interleukin-1 injection stimulated ACTH secretion in a dose-related manner, in both 2 and 24 month-old rats. Peak of the effect was 60 min after the injection. ACTH levels returned to baseline within 120 min in 2 month-old rats, whereas they were still high in untreated 24 month-old rats. However, plasma ACTH levels at 120 min were significantly lower in 24 month-old rats treated with pidotimod. Results suggest that pidotimod possesses immunomodulating properties, such as enhancement of splenocyte and peripheral blood lymphocyte proliferation, and improvement of the deficitary feedback mechanism between the hypothalamic-pituitary-adrenal axis and cytokines and, namely, interleukin-1 in the aging rat.

In vivo and in vitro effects of arginine-vasopressin receptor antagonists on the hypothalamic-pituitary-adrenal axis in the rat.

Arginine-vasopressin (AVP) is regarded as a potent stimulator of pituitary adrenocorticotropin (ACTH) secretion and participates therefore in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis function in concert with the physiological activator of the axis, hypothalamic corticotropin-releasing hormone (CRH). We examined the effects of AVP and/or three synthetic V1b receptor antagonists on the activity of the HPA axis in vivo and in vitro in the rat. AVP was injected intravenously to Sprague-Dawley rats (1 microgram/rat) through an indwelling jugular catheter. AVP stimulated ACTH release, with maximal effect 10 min after injection. Intravenous injection of three V1b antagonists, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-ethyltyrosine, 4-valine] arginine vasopressin (d(CH2)5[Tyr(Et2)]VAVP (WK 1-1), 9-desglycine[1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-ethyltyrosine, 4-valine] arginine vasopressin desGly9d(CH2)5 [Tyr(Et2)]-VAVP (WK 3-6), and 9-desglycine [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid),2-D-(O-ethyl)tyrosine, 4-valine ] arginine vasopressin des Gly9d(CH2)5[D-Tyr(Et2)]VAVP (AO 3-21), prevented AVP-stimulated ACTH secretion. Explanted rat hypothalami incubated in vitro with graded concentrations of AVP (10(-14)-10(-5) M) secreted immunoreactive CRH (iCRH) in a concentration-dependent fashion. Maximal stimulatory effect occurred at the concentration of 10(-6) M. Incubation of hypothalami with WK 1-1, WK3-6, or AO 3-21 (10(-6) M) prevented AVP-stimulated iCRH secretion. Results suggest that AVP plays a relevant, multiple role in the activation of the HPA axis in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulating effects of the synthetic thymic dipeptide pidotimod on the immune system in the aging rat.

We have investigated the in vivo and in vitro effect of pidotimod, a synthetic thymic-derived drug, on the immune response in young (2 month-old), and aging (24 month-old) Sprague-Dawley rats. We studied the effect of different doses of pidotimod on the responsiveness of both cultured peripheral blood lymphocytes and splenocytes to mitogenic stimuli, as well as on interleukin-2 production by peripheral blood lymphocytes after stimulation with interleukin-1 and phytohemagglutinin. Treatment with pidotimod in vivo as well as in vitro resulted in an increase of tritiated thymidine incorporation in both mitogen-stimulated lymphocytes and splenocytes from 24 month-old rats. Production of IL-2 from lymphocytes of 24 month-old rats was significantly increased in groups of animals treated with pidotimod. On the other hand, treatment with pidotimod did not influence the responsiveness of 2 month-old rat lymphocytes to mitogens, nor affected IL-2 production. Our results suggest a possible specific modulatory activity of pidotimod on the aging immune system.

Neurotransmitter-induced hypothalamic-pituitary-adrenal axis responsiveness is defective in inflammatory disease-susceptible Lewis rats: in vivo and in vitro studies suggesting globally defective hypothalamic secretion of corticotropin-releasing hormone.

The susceptibility of female Lewis (LEW/N) rats to the development of streptococcal cell wall (SCW)-induced arthritis and other autoimmune phenomena is associated with the inability of their hypothalamic-pituitary-adrenal (HPA) axis to adequately respond to inflammatory stimuli. In contrast, resistance to the development of SCW-induced arthritis and other inflammatory autoimmune manifestations in histocompatible female Fischer rats (F344/N) is related to their intact HPA axis response to inflammatory mediators. To evaluate the mechanism and the specificity of the HPA axis defect in LEW/N rats, we examined the ability of three major excitatory neurotransmitter systems to activate the HPA axis in both Lewis and Fisher rats. The responsiveness of plasma ACTH and corticosterone to the cholinergic muscarinic receptor agonist arecoline, the alpha 1-adrenergic receptor agonist methoxamine and the serotonin (5-HT) type 2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane were significantly blunted and/or abolished in LEW/N compared to F344/N rats. To localize the HPA axis defect to the hypothalamic CRH neuron, we evaluated the ability of explanted hypothalami from the two strains to secrete immunoreactive CRH in vitro, in response to acetylcholine (ACh), norepinephrine (NE), 5-HT and the 5-HT agonist quipazine. LEW/N hypothalami released less immunoreactive CRH (iCRH) in response to ACh, NE, 5-HT and quipazine than F344/N hypothalami. The dose-response curves of these compounds in the former were shifted to the right and/or abolished, suggesting decreased sensitivity of LEW/N hypothalami to these neurotransmitters.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between tumor necrosis factor-alpha, hypothalamic corticotropin-releasing hormone, and adrenocorticotropin secretion in the rat.

We studied the effects of tumor necrosis factor-alpha (TNF alpha), a macrophage-derived pleiotropic cytokine produced during the inflammatory/immune response, on the function of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Intravenous injections of TNF alpha stimulated plasma ACTH and corticosterone secretion in a dose-dependent fashion. This effect was inhibited by a rat CRH antiserum that was administered to the rats 1 h before the TNF alpha injections. This suggested that CRH is a major mediator of the HPA axis response to TNF alpha. We subsequently evaluated the ability of TNF alpha to influence CRH and ACTH secretion in vitro by explanted rat hypothalami in organ culture and by dispersed rat anterior pituicytes in primary culture respectively. Hypothalami were incubated for 40 min with graded concentrations of TNF alpha (10 pM to 1 microM). This cytokine stimulated CRH secretion in a dose-dependent fashion, with an EC50 of 6.7 x 10 pM (P less than 0.05). Preincubation of hypothalamic explants with dexamethasone, indomethacin (1 microM), eicosatetraynoic acid (10 microM), or nordihydroguaiaretic acid (30 microM) resulted in inhibition of TNF alpha-stimulated CRH secretion (P less than 0.05). Interestingly, 4-h incubation with TNF alpha had no effect on ACTH secretion from rat anterior pituicytes at a concentration of 10 nM. Higher concentrations of TNF alpha (100 nM and 1 microM), however, elicited a dose-dependent increase in the ACTH concentration in the medium. Our results suggest that TNF alpha represents one of the immune response mediators that directly or via stimulation of other cytokines act as activators of the HPA axis during immune/inflammatory reactions. This effect appears to be glucocorticoid suppressible and eicosanoid mediated. The primary site of action of TNF alpha appears to by the hypothalamic CRH-secreting neuron. Some pituitary and adrenal effects of TNF alpha, however, cannot be excluded.

Rat hypothalamic corticotropin-releasing hormone secretion in vitro is stimulated by interleukin-1 in an eicosanoid-dependent manner.

We studied the effect of interleukin-1 alpha (IL-1) on corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami in vitro. We also assessed possible mediation of arachidonic acid metabolites on IL-1-stimulated CRH secretion, by preincubating hypothalami with the cyclooxygenase inhibitor indomethacin (INDO, 1 microM), the lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid (ETYA, 10 microM), or the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, up to 30 microM). In additional experiments, prostaglandins (PG) E2 and F2 alpha were added to the cultures treated with INDO or ETYA. Finally, we investigated the effect of dexamethasone (DEX) on IL-1-stimulated CRH secretion. IL-1 stimulated immunoreactive CRH (iCRH) secretion by explanted hypothalami in a concentration-dependent fashion. Both INDO and ETYA inhibited IL-1-(10nM)-stimulated iCRH secretion, whereas NDGA did not have any effect. The addition of PGF2 alpha (10 nM) restored the secretion of iCRH inhibited by INDO. DEX treatment significantly inhibited IL-1-stimulated iCRH release. Our results suggest that the stimulatory effect of IL-1 on the hypothalamic CRH neuron is mediated by the cyclooxygenase metabolites of arachidonic acid, and, among others, by PGF2 alpha.

Procaine and lidocaine stimulate corticotropin-releasing hormone secretion by explanted rat hypothalami through a sodium conductance-independent mechanism.

We have previously shown that procaine and lidocaine stimulate corticotropin-releasing hormone (CRH) secretion by explanted rat hypothalami. This effect was of interest in light of the fact that both lidocaine and CRH administration to experimental animals can produce kindled seizures which cross-sensitize with electrically kindled seizures, and of recent data suggesting that limbic hyperexcitability, perhaps mediated through CRH, may be involved in the pathophysiology of affective illness. Because a prominent effect of the local anesthetics is to decrease neuronal firing by blocking sodium conductance, we were surprised by the capacity of these agents to cause CRH secretion and pituitary-adrenal activation and wished to further elucidate the possible mechanism(s) of these effects. To accomplish this, we first explored the effect of the sodium channel blocker tetrodotoxin (TTX) on basal and stimulated immunoreactive CRH (iCRH) secretion by explanted rat hypothalami. In contrast to procaine and lidocaine, TTX inhibited rather than stimulated iCRH secretion. Moreover, TTX inhibited lidocaine-induced iCRH secretion but had no influence on the response of the CRH neuron to procaine. To explore other potential mechanisms of action, we examined the effect of the calcium channels blocker verapamil and of pharmacologic antagonists to serotonergic, alpha-adrenergic and cholinergic receptors. The latter was particularly of interest because of structural similarities between procaine or lidocaine and acetylcholine (ACh) and because it has been shown that these anesthetic agents interact with the ACh receptor. Verapamil and blockade of serotonergic, alpha-adrenergic and cholinergic receptors did not inhibit the effects of procaine or lidocaine on iCRH secretion, whereas both GABA and dexamethasone exerted inhibitory effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid metabolites modulate rat hypothalamic corticotropin-releasing hormone secretion in vitro.

Arachidonic acid metabolites have been shown to modulate the secretion of various hormones, including luteinizing hormone, growth hormone and adrenocorticotropin. In this paper we describe the effect of a series of eicosanoids on hypothalamic secretion of corticotropin-releasing hormone (CRH) in vitro. Explanted rat hypothalami in culture were exposed to prostaglandins (PG) F2 alpha or E2, thromboxane (TX) B2, the TXA2 receptor agonist U-49,619 and leukotrienes (LT) B4, C4 and D4 at concentrations ranging from 10(-15) to 10(-5) M. PGE2, LTD4 and TXB2 did not alter hypothalamic CRH secretion. On the other hand, the remaining eicosanoids tested induced a significant increase of hypothalamic CRH secretion (p less than 0.05). The concentration of 10(-11) M dexamethasone inhibited the effect of stimulatory eicosanoids on CRH secretion. The CRH response to U-49,619 was completely prevented by the TXA2 receptor antagonist SQ-29,548. The latter also inhibited serotonin (5-HT)-, acetylcholine (ACh)- and PGF2 alpha-induced CRH release. Indomethacin was capable of blocking the secretion of CRH induced by 5-HT and ACh. In addition, PGE2 inhibited the increase of CRH secretion induced by PGF2 alpha, 5-HT and ACh. These findings suggest that eicosanoids may be involved in the regulation of hypothalamic CRH secretion, either as autocrine/paracrine or as endocrine factors.

[Importance of a particular formula of soybean milk without S2 globulin, in the treatment of allergy to cow's milk proteins]. / Importanza di una particolare formula di latte di soia priva della globulina S2, nel trattamento dell'allergia alle proteine del latte vaccino (APLV).

Replacing cow's milk with a soybean milk is one of the first measures which are commonly taken with babies suffering from cow's milk protein allergy. Unfortunately soybean can induce sensitization, too, with a variable percentage (ranging from 12 to 80%) according to the different Authors, due to the presence of allergenic proteins, among which the most important one seems to be S2 globulin. An excellent tolerability has been reported in a study carried out with 20 babies suffering from cow's milk protein allergy besides a good palatability of the product with a solution of symptoms in 87.3% of the cases. Only in 16.7% of the treated babies intolerance has been reported and therefore we can conclude that soybean milk has proved to be an excellent substitute of cow's milk in babies suffering from cow's milk protein allergy.

The alkyl-ether phospholipid platelet-activating factor is a stimulator of the hypothalamic-pituitary-adrenal axis in the rat.

Platelet-activating factor (PAF) is a naturally occurring alkyl-ether phospholipid which serves as an extracellular mediator in various cellular processes. Here we examined the effects of PAF on the activity of the hypothalamic-pituitary-adrenal axis in vivo and in vitro. PAF injected iv to rats (125, 250, and 500 ng/100 g BW) caused significant stimulation of pituitary ACTH and adrenal corticosterone secretion. The peak of PAF effect was recorded 10 min after the injection. Intraperitoneal injection of the PAF receptor antagonist BN 52021 prevented the ACTH-releasing effect of PAF. In explanted rat hypothalami maintained viable in vitro, PAF stimulated immunoreactive CRH secretion in a bell-shaped dose-response fashion. The maximal stimulatory effect occurred at the concentration of 10 nM. Higher concentrations appeared to cause desensitization. Alprazolam (1 microM) and BN 52021 (1 microM), two structurally different PAF receptor antagonists, inhibited this effect. The inhibitors of arachidonic acid metabolism, indomethacin, eicosatetraynoic acid, and the calcium channel blocker verapamil, inhibited PAF-stimulated CRH secretion, suggesting mediation by Ca2+ influx and phospholipase-A2 activation. In addition, we found that 1 nM PAF weakly stimulated ACTH secretion by dispersed rat pituicytes. This stimulatory effect of PAF was also inhibited by the receptor antagonists alprazolam and BN 52021. Our data suggest that PAF plays a role in the activation of the hypothalamic-pituitary-adrenal axis and glucocorticoid secretion and can perhaps serve as a mediator in the interactions of the immune system with the central nervous system.

A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats.

We have recently found that susceptibility to streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats is due, in part, to defective inflammatory and stress mediator-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis. Conversely, the relative arthritis resistance of histocompatible Fischer (F344/N) rats is related to their intact responses to the same stimuli. Specifically, LEW/N rats, in contrast to F344/N rats, have markedly impaired plasma corticotropin and corticosterone responses to SCW, recombinant human interleukin 1 alpha, the serotonin agonist quipazine, or synthetic rat/human corticotropin-releasing hormone (CRH). To explore the mechanism of this defect, we examined the functional integrity of the hypothalamic CRH neuron in LEW/N rats compared to F344/N rats. LEW/N rats, in contrast to F344/N rats, showed profoundly deficient paraventricular nucleus CRH mRNA levels and hypothalamic CRH content in response to SCW. Compared to F344/N rats, there was no increase in LEW/N hypothalamic CRH content or CRH release from explanted LEW/N hypothalami in organ culture in response to recombinant interleukin 1 alpha. These data provide strong evidence that the defective LEW/N corticotropin and corticosterone responses to inflammatory and other stress mediators, and the LEW/N susceptibility to experimental arthritis, are due in part to a hypothalamic defect in the synthesis and secretion of CRH. The additional finding of deficient expression in LEW/N rats of the hypothalamic enkephalin gene, which is coordinately regulated with the CRH gene in response to stress, suggests that the primary defect is not in the CRH gene but is instead related to its inappropriate regulation.

Effects of serotonergic agonists and antagonists on corticotropin-releasing hormone secretion by explanted rat hypothalami.

Experimental evidence suggests that serotonin (5HT) is excitatory to the hypothalamic-pituitary-adrenal axis and that this effect involves activation of both hypothalamic corticotropin-releasing hormone (CRH) and pituitary ACTH secretion. The present study was undertaken to examine the mechanism by which 5HT stimulates the central component of the HPA axis. To accomplish this we employed an in vitro rat hypothalamic organ culture system in which CRH secretion from single explanted hypothalami was measured by specific radioimmunoassay (IR-rCRH). All experiments were performed after an overnight (15-18 hr) preincubation. Serotonin stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped and the peak effect was observed at the concentration of 10(-9) M. The stimulatory effect of 10(-9) M 5HT was antagonized by the 5HT1 and 5HT2 receptor metergoline and by the selective 5HT2 receptor antagonists ketanserin and ritanserin. The muscarinic antagonist atropine, the nicotinic antagonist hexamethonium and the alpha-adrenergic receptor antagonist phentolamine, on the other hand, did not inhibit 5HT-induced IR-rCRH secretion. The specific 5HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) stimulated IR-rCRH secretion in a dose-dependent fashion. The response was bell-shaped with peak of effect reached at the concentration of 10(-9) M. We also tested the ability of the 5HT agonist meta-chlorophenylpiperazine (m-CPP) and of the selective 5HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) to cause CRH secretion. Although both m-CPP and 8-OH-DPAT stimulated IR-rCRH secretion in a dose-dependent fashion, several differences were observed when their effect was compared to that of 5HT. These included a different shape of the dose-response curve, a lower maximal stimulatory effect and a different maximal stimulatory concentration. These findings suggest that serotonin stimulates CRH secretion by explanted rat hypothalami and that this effect appears to be mediated mainly through a 5HT2 receptor mechanism.

Effect of cholinergic agonists and antagonists on rat hypothalamic corticotropin-releasing hormone secretion in vitro.

Several lines of experimental evidence suggest that acetylcholine (ACh) is excitatory to the hypothalamic-pituitary-adrenal (HPA) axis. Since previous experiments have shown that ACh does not affect pituitary adrenocorticotropin secretion in vitro, we hypothesized that ACh stimulates the HPA axis by causing hypothalamic corticotropin-releasing hormone (CRH) secretion. We examined this hypothesis using an organ culture system that measures the ability of single rat hypothalami to secrete immunoreactive CRH (IR-rCRH) in vitro. ACh stimulated hypothalamic IR-rCRH secretion in a dose-dependent fashion, at concentrations ranging from 3.3 x 10(-10) to 10(-5) M. This effect was antagonized by the simultaneous presence of atropine and hexamethonium, a muscarinic and a nicotinic receptor antagonist, respectively (p less than 0.05). Further evidence for the cholinergic regulation of the CRH neuron was provided by the findings that both carbachol, a muscarinic receptor agonist, and nicotine, a nicotinic receptor agonist, stimulated IR-rCRH secretion in a dose-dependent fashion. These effects were antagonized by atropine and hexamethonium, respectively, suggesting that both muscarinic and nicotinic receptors are involved in the process. ACh stimulated hypothalamic IR-rCRH secretion in the presence of phentolamine, an alpha-adrenergic antagonist, and ritanserin, a serotonin2 receptor antagonist, suggesting that the cholinergic stimulation of CRH secretion is not mediated by alpha-adrenergic or serotonergic interneurons. We conclude that ACh stimulates hypothalamic CRH secretion via both muscarinic and nicotinic receptor mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of rat hypothalamic corticotropin-releasing hormone secretion in vitro: potential clinical implications.

In summary, 5HT, ACh, NE, E and DA appear to stimulate hypothalamic CRH secretion whereas activation of the GABA/BZD system seems to decrease the responsivity of the CRH neuron to stimulatory neurotransmitters (Fig. 6). Hypothalamic CRH released from the hypothalamic neuron not only activates the HPA axis, but also stimulates the locus coeruleus-norepinephrine system (LC) and the central sympathetic system (CSS). CRH also induces secretion of hypothalamic POMC gene-derived peptides, such as ACTH, beta-EP, alpha-MSH and CLIP. These peptides as well as CRH itself, decrease the responsivity of the CRH neuron to stimulatory inputs. In addition, glucocorticoids restrain the activity of both the CRH neuron and the locus coeruleus and may also inhibit the secretion of POMC gene-derived peptides by the POMC neurons of the arcuate nucleus. Hypothalamic CRH secretion is regulated also by a number of mediators of the immune response, such as IL-1, IL-2, TNF-alpha and PGF2 alpha, PAF and EGF. Although the physiologic significance of this regulation is largely unknown, it is tempting to speculate that cytokines and mediators of inflammation released in vivo may activate the HPA axis to trigger a glucocorticoid-mediated counter-regulatory mechanism to restrain the immune system (Fig. 7). (Formula: see text). Fig. 7. Schematic representation of the interactions between the HPA axis and the immune system. Continuous lines represent stimulatory inputs and interrupted lines represent inhibitory inputs. In conclusion, our in vitro hypothalamic organ culture system allowed us to examine the regulation of CRH secretion in a direct and specific manner. Some of our observations may help with better understanding of the role played by CRH in the complex symptomatology of stress. In making extrapolations and interpretations from the in vitro data, however, we should try to keep in mind the words of Claude Bernard, "... If we break up a living organism by isolating its different parts it is only for the sake of ease in analysis and by no means in order to consider them separately. Indeed when we wish to ascribe to a physiological quality its value and true significance we must always refer it to this whole and draw our final conclusions only in relation to the effects in the whole".