The remodelled tracheal basement membrane zone of infant rhesus monkeys after 6 months of recovery

BACKGROUND: In previous studies, we showed that repeated exposure to (1) house dust mite allergen (HDMA) (Dermatophagoides farinae) caused thickening of the basement membrane zone (BMZ) and (2) HDMA+ozone (O3) caused depletion of BMZ perlecan and atypical development of BMZ collagen (irregular thin areas<2.0 microm in width). OBJECTIVE: The purpose of this study was to determine if these remodelling changes were reversible after 6 months of recovery. METHODS: Rhesus monkeys were exposed to a regimen of HDMA and or O3 or filtered air (FA) for 6 months. After the exposure protocol was completed FA and O3 groups were allowed to recover in FA for 6 months. The HDMA and HDMA+O3 exposure groups recovered in a modified environment. They were re-exposed to HDMA aerosol for 2 h at monthly intervals during recovery in order to maintain sensitization for pulmonary function testing. To detect structural changes in the BMZ, collagen I and perlecan immunoreactivity were measured and compared to data from the previous papers. RESULTS: The remodelled HDMA group had a significantly thicker BMZ and after 6 months of recovery the width had not regressed. In the remodelled BMZ of the HDMA+O3 group, perlecan had returned to the BMZ after 6 months of the recovery protocol, and the thin, irregular, collagen BMZ had been resolved. CONCLUSION: In summary, this study has shown that: (1) The width of the remodelled HDMA BMZ did not regress during a recovery protocol that included a sensitizing dose of HDMA. (2) The atypical collagen BMZ in the HDMA+O3 BMZ was resolved in the absence of O3. (3) Depletion of perlecan from the BMZ by O3 was reversed by recovery in the absence of O3.     

Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen

BACKGROUND: Airway smooth muscle hypertrophy is closely associated with the pathophysiology of hyper-reactive airways in allergic asthma. OBJECTIVE: To determine whether repeated exposure to allergens during postnatal lung development promotes remodelling of airway smooth muscle. METHODS: Infant, male rhesus monkeys (30-day-old) were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol periodically over 5 months. Smooth muscle mass and bundle size and abundance in conducting airways were measured and compared with age-matched control (filtered air-exposed) monkeys. RESULTS: Total smooth muscle mass and average bundle size were significantly greater in the conducting airways of monkeys exposed to HDMA. Smooth muscle bundle abundance was not affected by exposure to HDMA. CONCLUSION: Repeated cycles of allergen exposure alter postnatal morphogenesis of smooth muscle, affecting both total mass and bundle size, in conducting airways of infant monkeys.     

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae)

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.     

Early postnatal exposure to allergen and ozone leads to hyperinnervation of the pulmonary epithelium

Airway injury in infant monkeys exposed to ozone and/or house dust mite allergen (HDMA) is associated with a loss of epithelial innervation. In this study, we evaluated for persistence/recovery of the altered epithelial innervation. Thirty-day-old rhesus monkeys were exposed to repeated episodes of HDMA and/or ozone from 1 to 6 months of age and subsequently allowed to recover for 6 months in the absence of further ozone exposure and/or minimal HDMA challenge (sufficient to maintain allergen sensitization). At 1 year of age, nerve density in intrapulmonary airways was immunohistochemically evaluated using antibodies directed against protein gene product 9.5. Hyperinnervation and irregular epithelial nerve distribution was observed in both HDMA- and ozone-exposed groups; most prominent alterations were observed in animals exposed to HDMA plus ozone. Therefore, while adaptive mechanisms exist that re-establish epithelial innervation following cessation or diminution of exposure to HDMA and/or ozone, the recovery is associated with persistent proliferative mechanisms that result in hyperinnervation of the airways.     

Postnatal Development of the Lamina Reticularis in Primate Airways

The basement membrane zone (BMZ) appears as three component layers: the lamina lucida, lamina densa, and lamina reticularis. The laminas lucida and densa are present during all stages of development. The lamina reticularis appears during postnatal development. Collagens I, III, and V form heterogeneous fibers that account for the thickness of the lamina reticularis. Additionally, there are three proteoglycans considered as integral components of the BMZ: perlecan, collagen XVIII, and bamacan. Perlecan is the predominant heparan sulfate proteoglycan in the airway BMZ. It is responsible for many of the functions attributed to the BMZ, in particular, trafficking of growth factors and cytokines between epithelial and mesenchymal cells. Growth factor binding sites on perlecan include FGF‐1, FGF‐2, FGF‐7, FGF‐10, PDGF, HGF, HB‐EGF, VEGF, and TGF‐β. Growth factors pass through the BMZ when moving between the epithelial and mesenchymal cell layers. They move by rapid reversible binding with sites on both the heparan sulfate chains and core protein of perlecan. In this manner, perlecan regulates movement of growth factors between tissues. Another function of the BMZ is storage and regulation of FGF‐2. FGF‐2 has been shown to be involved with normal growth and thickening of the BMZ. Thickening of the BMZ is a feature of airway remodeling in asthma. It may have a positive effect by protecting against airway narrowing and air trapping. Conversely, it may have a negative effect by influencing trafficking of growth factors in the epithelial mesenchymal trophic unit. However, currently the significance of BMZ thickening is not known. Anat Rec, 293:947–954, 2008. © 2008 Wiley‐Liss, Inc.     

Asthma/allergic airways disease: does postnatal exposure to environmental toxicants promote airway pathobiology?

The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O3), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O3, or HDMA plus O3. The following alterations in the EMTU were found after exposure to HDMA, O3, or HDMA plus O3: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system. Conclusions: cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.     

Fluctuations in basal levels and effects of altered nutrition on plasma somatostatin

Rapid oscillations in basal plasma levels of insulin, C-peptide, glucagon, and glucose in rhesus monkeys have been reported previously. We now report large minute-to-minute fluctuations in plasma somatostatinlike immunoreactivity (SLI) in undisturbed, chair-adapted, and chronically cannulated male rhesus monkeys. Plasma samples were drawn from eight monkeys at 1- to 2-min intervals for 30-40 min following an overnight fast. Large fluctuations in SLI levels with periods of 4.4-9.4 min/cycle and possibly secondary periods of 14-18 min/cycle with average amplitudes of +/- 23% relative to the respective mean SLI levels were observed both within a single experiment and across monkeys under basal conditions. No consistent relationship was found between SLI fluctuations and that of insulin or glucagon. When portal and central blood samples were drawn precisely simultaneously no consistent portal-central gradient was found. Patterns of fluctuations in central and portal SLI levels were not correlated. Overfeeding significantly increased insulin levels but did not alter SLI levels. After 5 days of food deprivation basal SLI levels were unchanged. These data indicate the need to exercise care in the interpretation of single samples in the measurement of plasma SLI levels. Somatostatin secreted into the blood under basal, unstimulated conditions appears to be unrelated to nutritional balance.     

Novel extracellular matrix structures in the neural stem cell niche capture the neurogenic factor fibroblast growth factor 2 from the extracellular milieu

The novel extracellular matrix structures called fractones are found in the lateral ventricle walls, the principal adult brain stem cell niche. By electron microscopy, fractones were shown to contact neural stem and progenitor cells (NSPC), suggesting a role in neurogenesis. Here, we investigated spatial relationships between proliferating NSPC and fractones and identified basic components and the first function of fractones. Using bromodeoxyuridine (BrdU) for birth-dating cells in the adult mouse lateral ventricle wall, we found most mitotic cells next to fractones, although some cells emerged next to capillaries. Like capillary basement membranes, fractones were immunoreactive for laminin beta1 and gamma1, collagen IV, nidogen, and perlecan, but not laminin-alpha1, in the adult rat, mouse, and human. Intriguingly, N-sulfate heparan sulfate proteoglycan (HSPG) immunoreactivity was restricted to fractone subpopulations and infrequent subependymal capillaries. Double immunolabel for BrdU and N-sulfate HSPG revealed preferential mitosis next to N-sulfate HSPG immunoreactive fractones. To determine whether N sulfate HSPG immunoreactivity within fractones reflects a potential for binding neurogenic growth factors, we identified biotinylated fibroblast growth factor 2 (FGF-2) binding sites in situ on frozen sections, and in vivo after intracerebroventricular injection of biotinylated FGF-2 in the adult rat or mouse. Both binding assays revealed biotinylated FGF-2 on fractone subpopulations and on infrequent subependymal capillaries. The binding of biotinylated FGF-2 was specific and dependent upon HSPG, as demonstrated in vitro and in vivo by inhibition with heparatinase and by the concomitant disappearance of N-sulfate HSPG immunoreactivity. These results strongly suggest that fractones promote growth factor activity in the neural stem cell niche.     

Peripheral glomerular basement membrane thickness in the normal and diabetic monkey

Diabetes of varying severity was induced in a colony of rhesus monkeys by streptozotocin injection or total pancreatectomy. Peripheral glomerular basement membrane thickness was determined using a reproducible and efficient procedure. Four groups of monkeys formed on the basis of sex, age, severity, and duration of treatment were analyzed. In control animals peripheral glomerular basement membrane thickness increased with age from 3 to 7 years, whereas monkeys aged 11 to 18 years had relatively constant measurements. Mean values did not differ between control and age-matched non-insulin-dependent streptozotocin-injected animals treated up to 13 years previously. Insulin-dependent streptozotocin-treated or pancreatectomized monkeys with a treatment duration of 10 to 27 months were not different from age-matched controls, whereas insulin-dependent monkeys treated 2 to 12.5 years earlier showed significantly increased peripheral glomerular basement membrane thickness. The peripheral glomerular basement membrane thickening observed in the insulin-dependent monkey is easily measured and is similar to changes observed in human diabetes mellitus.     

Effect of epidermal growth factor on the fetal development of the tracheobronchial secretory apparatus in rhesus monkey

Fetal rhesus monkeys were treated with recombinant human epidermal growth factor (EGF) to determine if EGF can induce maturation of the tracheobronchial secretory apparatus. At 75% of gestation, EGF was administered simultaneously into both the amniotic fluid and fetal abdominal cavity at an average dose of 66 micrograms/kg body weight, by each route over a 7-d period. At the end of the treatment period, the fetuses were delivered and either euthanized immediately or after maintenance on ventilatory support for 6 h. The lungs were removed, and the trachea and one lobe of the right lung was fixed and embedded for light microscopy. The left lung was lavaged with saline, and the collected fluid was used to quantify released secretory product. Secretory product was also measured in amniotic fluid as well as in situ on histologic sections of tracheal epithelium. When the tracheas of EGF-treated monkeys were examined, the epithelium was found to be taller and to contain a greater proportion of secretory cells and a smaller proportion of intermediate cells than the control group. However, there was no significant change in the total number of cells per millimeter of basal lamina or in the proportion of the epithelial population made up of basal or ciliated cells. There was more secretory product stored in the epithelium and submucosal glands and increased quantities of respiratory secretions in both lung lavage and amniotic fluid of EGF-treated monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term effects of differential early rearing in rhesus macaques: behavioral reactivity in adulthood

Adverse early experiences are associated with a range of deleterious health outcomes in humans, including higher risk for affective disorders. Studies using a long-standing model of nonhuman primate model of early adversity have demonstrated that nursery-reared (NR) monkeys exhibit alterations in multiple aspects of biobehavioral development; however, few studies have evaluated the persistence of socioaffective behavioral changes through adulthood. We evaluated the effects of early rearing experience on adult animals' response to a well-validated assessment of anxiety-like behavior, the human intruder paradigm (HIP). We tested 22 rhesus monkeys who were either nursery-reared (NR) or reared with their mothers (mother-reared; MR). NR monkeys were inhibited in their behavior compared to MR monkeys, with reduced locomotion and exploratory behaviors. NR animals showed a marginal increase in freezing. Together these findings demonstrate that the consequences of differential infant rearing experience on socioaffective behavior persist into adulthood, with evidence of greater inhibition in NR monkeys.     

Kinematics of reaching and implications for handedness in rhesus monkey infants

Kinematic studies of reaching in human infants using two-dimensional (2-D) and three-dimensional (3-D) recordings have complemented behavioral studies of infant handedness by providing additional evidence of early right asymmetries. Right hand reaches have been reported to be straighter and smoother than left hand reaches during the first year. Although reaching has been a popular measure of handedness in primates, there has been no systematic comparison of left and right hand reach kinematics. We investigated reaching in infant rhesus monkeys using the 2-D motion analysis software MaxTRAQ Lite+ (Innovision Systems). Linear mixed-effects models revealed that left hand reaches were smoother, but not straighter, than right hand reaches. An early left bias matches previous findings of a left hand preference for reaching in adult rhesus monkeys. Additional work using this kind of kinematic approach will extend our understanding of primate handedness beyond traditional studies measuring only frequency or bouts of hand use.     

Acidic fibroblast growth factor is progressively increased in the development of oesophageal glandular dysplasia and adenocarcinoma

AIMS: To determine by immunohistochemistry and amplification of cDNA the relationship between fibroblast growth factor (FGF) expression and progressive changes in Barrett's oesophagus associated with oesophageal adenocarcinoma (OA). The FGFs are potent mitogens that possess angiogenic properties and the capability to regulate growth and differentiation of various cell types. They have also been implicated in the development and progression of numerous solid tumours, including some carcinomas of the aerodigestive tract, such as nasopharyngeal carcinoma and pancreatic adenocarcinoma. MATERIAL AND RESULTS: We studied the expression of the two prototypic FGFs, acidic FGF (FGF-1) and basic FGF (FGF-2), in OA and OA precursor lesions, including intestinal metaplasia (IM), low-grade dysplasia (LGD) and high-grade dysplasia (HGD). Fresh tissue from 10 OAs and four associated HGDs was available for the determination of FGF-1 and FGF-2 mRNA expression accomplished by the PCR amplification of cDNA. Using immunohistochemistry, we studied the expression of the FGF-1 and FGF-2 proteins in archival, paraffin-embedded tissue that was available from 17 oesophageal resection specimens that included OAs and OA precursor lesions. As compared to gastric fundic mucosal controls, OAs and HGDs showed significantly enhanced expression of FGF-1 mRNA and protein. IMs and LGDs showed significantly lesser degrees of FGF-1 immunoreactivity that were not increased over controls. In contrast, both the overall percentage of FGF-2-reactive OAs and the overall FGF-2 protein expression, assessed using an immunoreactivity score, are comparable to FGF-2 expression in controls. CONCLUSIONS: It appears that FGF-2 is ubiquitously expressed in OA and in normal oesophageal and gastric mucosa while significant FGF-1 expression is essentially restricted to HGD and OA. Our data also suggest that FGF-1 is sequentially upregulated in the progression from metaplasia to dysplasia and adenocarcinoma.     

Oligodendrocyte-specific protein is encephalitogenic in rhesus macaques and induces specific demyelination of the optic nerve

Oligodendrocyte-specific protein (OSP) is a candidate autoantigen in the development of multiple sclerosis (MS). We evaluated the potential of OSP to induce EAE in rhesus monkeys, an out bred animal model for MS that is immunologically close to humans. Since OSP is a four-membrane spanning protein with highly hydrophobic regions, we synthesized recombinant proteins encompassing only the hydrophilic regions of human OSP (soluble (s)hOSP). Immunization with shOSP proteins induced clinical signs and histological features of optic neuritis in four out of ten rhesus monkeys. The development of clinical disease was associated with the presence of a strong cellular proliferative response to the immunizing shOSP protein. Analysis of the cellular responses in combination with neuropathological observations also indicates an important role for neutrophils in the disease process. Interestingly, all immunized monkeys developed antibody responses to OSP peptide 103-123, a B cell epitope previously identified in MS patients. These responses did not correlate with the development of clinical disease, but may have relevance as a biomarker for immunoreactivity towards OSP in myelin disorders. Our data demonstrate that in rhesus monkeys immune responses directed at OSP are encephalitogenic, leading to inflammatory responses throughout the central nervous system and to selective demyelination of the optic nerve.     

Relationships between luminance and visual acuity in the rhesus monkey

1. The ability of rhesus monkeys to detect the gap in Landolt ring test-objects that were presented against background luminances between 5 x 10(-5) cd/m(2) and 5 x 10(3) cd/m(2) was compared with similar human data.2. At high luminance-levels the acuity of human observers is slightly better than that of rhesus, but rhesus have better acuity at scotopic luminance-levels. Both species have distinct photopic and scotopic acuity functions that cross at 6 x 10(-3) cd/m(2).3. The threshold for light detection is estimated to be the same for both species when specified in quanta incident on the retina.4. It is concluded that the receptor and neural mechanisms that mediate visual-acuity function similarly in rhesus and man, and that the differences in acuity that were measured in the two species may be attributed to optical rather than to physiological factors.     

The qualitative evaluation of airway responses to immunologic and pharmacologic stimuli in rhesus monkeys.

This report describes the current status of a colony of rhesus monkeys composed of a group of animals with consistent asthmatic responses to Ascaris antigen challenge, a variable group and a negative group. The cumulative experience with the consistent group of 5 animals totals 144 months of observation with 86 positive respiratory responses to 86 aerosol challenges. Further studies compare rhesus airway responses to Ascaris antigen, anti-IgE, histamine, prostaglandin (PG)F2alpha, carbocholine, and physostigmine. We report that two abnormalities of pulmonary function which occur as a result of aerosol challenge, an increase in breathing frequency (f) and pulmonary resistance (PR), differ in degree of abnormality and time of onset following challenge with different agonists. These results indicate that the f and PR changes in response to these agonists are controlled by different physiologic mechanisms in rhesus monkeys. We suggest that the f changes may occur as a result of reflex afferent vagal stimulation and PR changes as a result of direct effect on smooth muscle receptors. The effect of histamine and PGF2alpha on the airway of rhesus monkeys more closely simulates the airway response to immunologic stimuli than does the effect of cholinergic type agonists.     

Age-related changes of the ciliary muscle in comparison with changes induced by treatment with prostaglandin F2 alpha. An ultrastructural study in rhesus and cynomolgus monkeys

The relationship between individual ciliary muscle cells and the surrounding connective tissue was studied in the eyes of three normal, young (3-4 years) cynomolgus monkeys (Macaca fascicularis), three aged (34-36 years) rhesus monkeys (Macaca mulatta) and seven young (3-7 years) cynomolgus monkeys topically treated with prostaglandin F2 alpha (PGF2 alpha) for 4-8 days. In normal eyes, collagen fibrils and microfibrils are in places in continuity with the muscle cells' basal lamina, which is connected to the cell membrane by fine fibrillous material. In old eyes, the basal lamina is markedly thickened, masking the connection of fibrils with the muscle cells' membrane. A distinctive finding in several muscle cells of old eyes are electronlucent clefts, 60-80 nm wide, between basal lamina and cell membrane, which are not transversed by fibrils or fibrillous material. The cell membrane of these muscle cells shows large folds filled with disarranged myofilaments. Additionally, these cells contain inclusion bodies consisting of concentrically arranged double membranes. Following treatment with PGF2 alpha, similar changes are seen in young animals, too. Here, the muscle cells have lost their connection to the extracellular fibrils due to a PGF2 alpha-induced lysis of extracellular material. Lack of attachment between basal lamina and altered muscle cells in aged eyes might indicate an involvement of the extracellular matrix in age-related changes of the individual ciliary muscle cells.     

Immune and cytokine/chemokine responses of PBMCs in rotavirus-infected rhesus infants and their significance in viral pathogenesis

The rotavirus (RV) is the most important causative agent of severe gastroenteritis in infants and children aged less than 5 years worldwide. However, the response and the roles of peripheral blood mononuclear cell (PBMC) in RV clearance have yet to be fully elucidated. In this study, we established the neonatal rhesus monkey model of RV infection with histopathological changes in the small intestine. Then, we investigated gene expression changes in PBMCs from the monkey model of RV infection. Similar pathways regulated in rhesus monkeys that received intragastric administration of the RV monkey SA11 strain (G3P[2]) and the human wild-type strain ZTR-68 (G1P[8]). Gene profiling showed differences in functional genes mainly associated with chemokine signaling pathways and cytokine-cytokine receptor interactions post RV infection. Transferrin and C-C motif chemokine ligand 23 (CCL23) gene expression were upregulated in PBMCs of monkeys when stimulated by simian and human RV strains. Monkeys infected with RV had an enhanced and prolonged inflammatory response that was associated with increased levels of CCL20, CCL23, and C-X-C motif chemokine ligand 1; while inhibition of major histocompatibility complex class I expression may be important for immune evasion by RV. The RV infection was also characterized by pathological changes in the small intestine with a cytokine and chemokine storm. This study identified the chemokine signaling pathway and immune response genes involved in RV infection in infant rhesus monkeys. The SA11 RV strain is more suitable for establishing a monkey diarrhea model than the ZTR-68 RV strain.     

笼养恒河猴血清天然抗体IgM的检测

采用流式细胞术连续监测 8只幼年猴自出生后 1月～ 5月天然抗体的演变 ,并检测 9只成年猴天然抗体IgM水平。幼年猴 1月龄前天然抗体IgM阳性细胞比例较低 (9 9%± 6 3% ) ,到 3月龄时可达成年猴的 4 0 % ,5月龄时接近成年水平。 9只成年猴天然抗体IgM阳性细胞比例变化幅度很大 ,从 94 9%到 2 6 3% (均数 77 3%± 2 1 5 % )。提示 3月龄前幼年猴可用作避免超急性排斥反应的受体模型。不管是成年猴还是幼年猴 ,被选择为实验受体进行移植研究时 ,应事先对其体内的天然抗体水平进行检测 ,以确保实验的可比性。     

Differential airway reactivity to carbachol and physostigmine sulfate in rhesus monkeys with and without reagin-mediated respiratory responses.

Bronchial airway reactivity to carbachol (CAR) and physostigmine (PHY) was individually measured in three rhesus monkeys with and three rhesus monkeys without reagin-mediated respiratory responses (RR). The three monkeys with reagin-mediated RR demonstrated bronchial hyperreactivity to CAR (p less than 0.25) but not PHY when compared with the three control monkeys. No correlation between airway reactivity to CAR and PHY was noted in all six monkeys. We conclude that monkeys with reagin-mediated RR possess airway hyperreactivity to the exogenously administered acetylcholine analogue CAR, but not to the endogenous acetylcholine which is released in vivo by PHY.