Vasointestinal Peptide Research Articles

PAR-2 modulates pepsinogen secretion from gastric-isolated chief cells.

In the present study, we investigated whether activation of protease-activated receptor type 2 (PAR-2) with SLIGRL (SL)NH2, a short mimetic agonistic peptide, directly stimulates pepsinogen secretion from gastric-isolated, pepsinogen-secreting (chief) cells. Immunostaining of gastric-dispersed chief cells with a specific anti-PAR-2 antibody demonstrated expression of PAR-2 receptors on membrane and cytoplasm. SL-NH2 and trypsin potently stimulated pepsinogen secretion (EC50 = 0.3 nM) and caused Ca2+ mobilization (EC50 = 0.6 nM). In contrast to SL-NH2, the scramble peptide LSIGRL-NH2 failed to stimulate pepsinogen release. Exposure to SL-NH2 also resulted in ERK1/2 phosphorylation and activation. Exposure of chief cells to phosphotyrosine kinase inhibitors and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a selective MEK inhibitor, significantly reduced secretion induced by SL-NH2. Pepsinogen secretion induced by SL-NH2 was desensitized by pretreating the cells with the mimetic peptide and trypsin, and exposure to SL-NH2 abrogates pepsinogen secretion induced by carbachol and CCK-8, but not secretion induced by secretin and vasointestinal peptide. Exposure to Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (substance P) but not to calcitonin gene-related peptide increased pepsinogen release. The neurokinin-1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester, inhibited substance P-stimulated pepsinogen secretion, whereas it did not affect secretion induced by SL-NH2. Collectively, these data indicate that PAR-2 is expressed on gastric chief cells and that its activation causes a Ca2+-ERK-dependent stimulation of pepsinogen secretion.

Pharmacological sensitivity and gene expression analysis of the tibial nerve injury model of neuropathic pain

The tibial nerve injury model is a novel, surgically uncomplicated, rat model of neuropathic pain based on a unilateral transection (neurotomy) of the tibial branch of the sciatic nerve. The aim of the present study was to describe some behavioral and molecular features of the model, and to test its sensitivity to a number of drugs which are currently used for the treatment of neuropathic pain. The model was characterized by a pronounced mechanical allodynia which was present in all subjects and a less robust thermal hyperalgesia. Mechanical allodynia developed within 2 weeks post-surgery and was reliably present for at least 9 weeks. Neurotomized rats showed no autotomy and their body weight developed normally. Gene expression in ipsilateral L5 dorsal root ganglia, analyzed by quantitative polymerase chain reaction (PCR), showed a pronounced up-regulation of galanin and vasointestinal peptide (VIP). This up-regulation developed rapidly (within 1 to 2 days following neurotomy) and remained present for at least 12 days. On the other hand, expression of calcitonin gene-related peptide (CGRP) and substance P mRNA was down-regulated 12 days following neurotomy. Mechanical allodynia was completely reversed by morphine [minimal effective dose (MED): 8 mg/kg, i.p.] and partially reversed by carbamazepine (MED: 64 mg/kg, i.p.), baclofen (MED: 3 mg/kg, i.p.) and amitriptyline (trend for efficacy at 32 mg/kg, i.p.), but not by gabapentin (50–100 mg/kg, i.p.). The finding that the tibial nerve injury model shows a robust and persistent mechanical allodynia which is sensitive to a number of established analgesics, as well as a gene expression profile which is compatible with that obtained in other models of neuropathic pain, further supports its validity as a reliable and surgically uncomplicated model for the study of neuropathic pain.

Pacemaker Neurons and Secreted Factors

Three papers this week provide new insights into the molecular mechanisms underlying the establishment and maintenance of circadian rhythms. Cyclic periods of activity and inactivity occur in the absence of changes in light, indicative of an autonomous clock. This cycle also is influenced by light. Nitabach et al. created transgenic flies in which electrical activity in the pacemaker neurons, which control circadian changes in locomotor activity, was suppressed by targeted expression of K+ channels. These flies did not exhibit circadian changes in activity in continued dark, nor did the pacemaker cells exhibit molecular oscillation of the Period and Timeless proteins in the dark. Light entrainment of the molecular clock was unaffected, suggesting that the electrical activity of the pacemaker neurons is only essential for molecular periodicity of these neurons in the absence of environmental clues. Two groups investigated the circadian clock in mice. Harmar et al. investigated the circadian clock of mice lacking the receptor VPAC2, which recognizes both vasointestinal peptide (VIP) and pituitary adenylate cyclase activating-polypeptide (PACAP) and which is abundant in the suprachiasmatic nuclei (SCN), the site of the mammalian master clock. The VPAC2-/- mice exhibited decreased circadian behavior patterns and more rapid reentrainment in response to changes in the light:dark cycle compared with wild-type animals. Furthermore, the rhythmic changes in the clock genes in the SCN were suppressed in the VPAC2-/- mice under conditions with a light and dark cycle, and rhythmicity was completely lost in the VPAC2-/- SCN in mice kept in constant dark. Cheng et al. provide evidence for prokineticin 2 as a secreted factor that may be a key output of the SCN clock activity in mammals. Prokineticin 2 is expressed in the SCN, the receptor for prokineticin 2 in regions of the brain that are targets of the SCN. The expression of prokineticin 2 is rhythmic and regulated in response to light. Finally, injection of prokineticin 2 into the brains of mice inhibited locomotor activity during the dark phase and increased activity during the light phase.M. N. Nitabach, J. Blau, T. C. Holmes, Electrical silencing of Drosophila pacemaker neurons stops the free-running cicradian clock. Cell 109, 485-495 (2002). [Online Journal]A. J. Harmar, H. M. Marston, S. Shen, C. Spratt, K. M. West, W. J. Sheward, C. F. Morrison, J. R. Dorin, H. D. Piggins, J.-C. Reubi, J. S. Kelly, E. S. Maywood, M. H. Hastings, The VPAC2 receptor is essential for cicadian function in the mouse suprachiasmatic nuclei. Cell 109, 497-508 (2002). [Online Journal]M. Y. Cheng, C. M. Bullock, C. Li, A. G. Lee, J. C. Bermak, J. Belluzzi, D. R. Weaver, F. M. Leslie, Q.-Y. Zhou, Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature 417, 405-410 (2002). [Online Journal]

Pacemaker Neurons and Secreted Factors

Three papers this week provide new insights into the molecular mechanisms underlying the establishment and maintenance of circadian rhythms. Cyclic periods of activity and inactivity occur in the absence of changes in light, indicative of an autonomous clock. This cycle also is influenced by light. Nitabach et al. created transgenic flies in which electrical activity in the pacemaker neurons, which control circadian changes in locomotor activity, was suppressed by targeted expression of K + channels. These flies did not exhibit circadian changes in activity in continued dark, nor did the pacemaker cells exhibit molecular oscillation of the Period and Timeless proteins in the dark. Light entrainment of the molecular clock was unaffected, suggesting that the electrical activity of the pacemaker neurons is only essential for molecular periodicity of these neurons in the absence of environmental clues. Two groups investigated the circadian clock in mice. Harmar et al. investigated the circadian clock of mice lacking the receptor VPAC 2 , which recognizes both vasointestinal peptide (VIP) and pituitary adenylate cyclase activating-polypeptide (PACAP) and which is abundant in the suprachiasmatic nuclei (SCN), the site of the mammalian master clock. The VPAC 2 -/- mice exhibited decreased circadian behavior patterns and more rapid reentrainment in response to changes in the light:dark cycle compared with wild-type animals. Furthermore, the rhythmic changes in the clock genes in the SCN were suppressed in the VPAC 2 -/- mice under conditions with a light and dark cycle, and rhythmicity was completely lost in the VPAC 2 -/- SCN in mice kept in constant dark. Cheng et al. provide evidence for prokineticin 2 as a secreted factor that may be a key output of the SCN clock activity in mammals. Prokineticin 2 is expressed in the SCN, the receptor for prokineticin 2 in regions of the brain that are targets of the SCN. The expression of prokineticin 2 is rhythmic and regulated in response to light. Finally, injection of prokineticin 2 into the brains of mice inhibited locomotor activity during the dark phase and increased activity during the light phase. M. N. Nitabach, J. Blau, T. C. Holmes, Electrical silencing of Drosophila pacemaker neurons stops the free-running cicradian clock. Cell 109 , 485-495 (2002). [Online Journal] A. J. Harmar, H. M. Marston, S. Shen, C. Spratt, K. M. West, W. J. Sheward, C. F. Morrison, J. R. Dorin, H. D. Piggins, J.-C. Reubi, J. S. Kelly, E. S. Maywood, M. H. Hastings, The VPAC 2 receptor is essential for cicadian function in the mouse suprachiasmatic nuclei. Cell 109 , 497-508 (2002). [Online Journal] M. Y. Cheng, C. M. Bullock, C. Li, A. G. Lee, J. C. Bermak, J. Belluzzi, D. R. Weaver, F. M. Leslie, Q.-Y. Zhou, Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus. Nature 417 , 405-410 (2002). [Online Journal]

Determination of changes in mRNA expression in a rat model of neuropathic pain by Taqman™ quantitative RT-PCR

The aim of this study was to develop a rapid and accurate high throughput method of screening multiple genes across a single sample set to detect changes in gene expression in the dorsal root ganglion (DRG) following partial sciatic nerve ligation in the rat. Using Taqman quantitative RT-PCR, we show that expression of a number of genes, including galanin, vasointestinal peptide and neuropeptide Y are rapidly increased 24 h post-operation in the DRGs on the ligated side only. Other genes tested, including vanilloid receptor-1, substance P, galanin receptor-2 and housekeeping genes did not alter. Analysis of the expression of ASIC4 showed a small difference in expression at 7 days post ligation. By applying a statistical method for analysis of multiple variables, partial least squares, we show that the expression change of ASIC4 was significantly altered on the ligated side even though the change was small. This method will allow us to rapidly identify changes in expression of candidate genes that may be involved in adaptive responses in the DRG due to nerve injury.

Retinoic acid treatment enhances the acetylcholine contents in the human teratocarcinoma cell line NTera-2

Human NTera-2/clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptide-contents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 μM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca ++ dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.

The biological basis of female sexuality.

Human sexuality has three main roots: biological, motivational-affective-relational, and cognitive. Unfortunately, in women, the biological dimension is usually disregarded. Hormones are necessary, but not sufficient, factors to maintain a satisfying human libido. In women, oestrogens prime the central nervous system, acting as neurotrophic and psychotrophic factors throughout life. They also prime the sensory organs, including the skin with its sebaceous and sweat glands, which are the key receptors for external sexual stimuli. Oestrogens are also the 'permitting factors' for the action of vaso-intestinal peptide, the key neurotransmitter involved in the endothelial and vascular changes leading to vaginal lubrication. Other factors, such as medication, alcohol and other health problems, can modify the biological impact of hormones on libido. Depression may cause a progressive decline in interest in sexual behaviour leading to low libido, difficulty in sexual arousal, secondary anorgasmia and/or frank sexual aversion. Increasing attention of doctors towards the sexual problems of women will dramatically improve female quality of life, especially during difficult periods of transition.

Radionuclide imaging of neuroendocrine tumours: biological basis and diagnostic results.

Neuroendocrine tumours have been defined as APUD-omas in the past by authors who identified common metabolic characteristics (amine precursor uptake and decarboxylation) in a group of tumours thought to originate from cells of the neural crest and to be able to produce biogenic amines. The identification of neuroendocrine tumours with APUD-omas was not confirmed by subsequent investigators. At present it is known that a group of neuroendocrine tumours derive from pluripotent stem cells or from differentiated neuroendocrine cells, and that they have a particular pattern of histology due to the presence of some secretory products and particular cytoplasmic proteins. Many radiopharmaceuticals have been successfully used in nuclear medicine to visualise neuroendocrine tumours; most of them are based on specific uptake mechanisms, but some are non-specific probes. This review is focussed on the clinical application of radiolabelled metaiodobenzylguanidine, indium-111 pentetreotide, radiolabelled vasointestinal peptide, radiolabelled monoclonal antibodies and positron-emitting tracers. While many different types of neuroendocrine tumours are identified today, only the most common histotypes and those tumours of major relevance for nuclear medicine are considered in this review (anterior pituitary tumours and neuroblastoma are excluded). New knowledge in molecular biology, relevant biological and histological patterns, and the physiological and clinical behaviour are described for neuroendocrine tumours of the lung, tumours of the gastroenteropancreatic tract, medullary thyroid carcinoma, tumours of sympatho-adrenal lineage, and multiple endocrine neoplasia. The nuclear medicine results in diagnostic imaging are presented, and the major comparative studies with different tracers are reported. The study of further possible diagnostic approaches addressing the biological characteristics of these tumours could open the way to various new therapeutic options.

Mack Forster Award Lecture. Receptor nuclear medicine: vasointestinal peptide and somatostatin receptor scintigraphy for diagnosis and treatment of tumour patients.

The multiple aspects of radioligand-receptor interactions do not only show a major impact of certain cell surface-bound receptors in the pathophysiology of human disease: the concept of radioligand-receptor interactions has also been extended to the clinic. In particular, naturally occurring peptides, when radiolabelled, are clinically useful for the imaging diagnosis of human disease and have future implications for the treatment of tumour expressing certain target receptors using radiolabelled peptide tracers. The finding that receptors for VIP (vasoactive intestinal peptide) and SST (somatostatin) are overexpressed on tumour cells presents a breakthrough into this direction. Recent data indicate that [123I]-VIP receptor scintigraphy is clinically useful for the in vivo localization of small primary adenocarcinomas, liver metastases and certain endocrine tumours of the gastrointestinal tract. After the successful clinical introduction of the SST analogues [123I]-Tyr3-octreotide and [111In]-DTPA-D-Phe1-octreotide for localization diagnosis of neuroendocrine tumours in 1989, P829, labelled with the more cost-effective radionuclide 99mTc, nowadays promises to be a potential novel diagnostic imaging agent for tumours expressing SST/VIP receptors. Furthermore, the novel SST analogue [90Y]-MAURITIUS is entering the clinic for treatment of VIP/SST receptor-expressing tumours.

Environmental and genetic determinants of neural migration and postmigratory survival.

The study of genetic/epigenetic/environmental factors underlies all therapeutic and preventive approaches in fetal, perinatal and paediatric neurology, including rehabilitation. In this paper, we selected a few targets of environmental determinants of brain development leading to underlying priorities for protection of the developing brain. Preparation of the neural germinative epithelium has to be protected against noxious pharmacological agents. New tools have been developed to improve early neural teratology, including the whole-embryo culture method. The neopallial astrocytic precursors have a dual origin. Astrocytes of the white matter and deep neocortical layers derive from transformed radial glial cells, whereas astrocytes of the upper neocortical layers derive from astrocytic precursors that migrate from the late germinative zone after the end of neuronal migration. Among numerous factors able to interfere with these gliogenetic events are the control factors of the lysosomal and autophagic functions, interfering with radial glial cell transformation into astrocytes. All lesions interrupting the migratory corridors of late astroglial migration can produce cytoarchitectonic disturbances of the neocortical supragranular layers, with long-term consequences. The developing brain is weltering in a complex mixture including newly recognized excitotoxic substances, cytokines and growth factors. These substances are sometimes environmental friends like maternal vasointestinal peptide, which prevents brain intrauterine growth retardation. They are sometimes excellent endogenous friends like neurotrophic excitatory agents in physiological conditions. They become often dangerous killers triggered by environmental signals like hypoxias/ischaemias and toxins produced by intrauterine infections, launching the excitotoxic cascade. In this paper, we reviewed mainly environmental determinants interfering with neural cytogenesis and histogenesis during the embryonic, fetal and neonatal span of early life.

Release of internal CA2+ and activation of constitutive nitric oxide synthase (cNOS) by vasointestinal peptide (VIP) in endothelial cells (EC)

Release of internal CA2+ and activation of constitutive nitric oxide synthase (cNOS) by vasointestinal peptide (VIP) in endothelial cells (EC)

Nasal Pathology and infrastructure in Patients with Chronic Airway Inflammation (RADS and RUDS) Following an Irritant Exposure

Reactive airways dysfunction syndrome is a chronic asthma-like condition developing after an acute irritant exposure, and chronic inflammation has been seen on endobronchial biopsy. Reactive upper-airways dysfunction syndrome is chronic rhinitis developing in temporal association with a toxic inhalation exposure, but the pathophysiology is unknown. To study biopsies of the nasal mucosa in patients with reactive upper-airways dysfunction syndrome and in some cases reactive airways dysfunction syndrome developing in temporal association with a chlorine dioxide exposure, to see if a histologic basis for the persistent rhinitis and sensitivity to chemical irritants could be determined. Specimens were stained with hematoxylin-eosin and immunoperoxidase stains for substance P, vasointestinal peptide, and S-100 (nerve fibers), and fixed in glutaraldehyde for electron microscopy. Biopsies of three nonexposed subjects were performed for comparison. A pathologist blinded to clinical data interpreted the specimens. Inflammation ratings of exposed individuals were higher than for the nonexposed individuals. The number of nerve fibers stained was greater for patients vs controls. Substance P and vasointestinal peptide staining was nonspecific. Electron microscopy showed desquamation of the epithelium and permeability of epithelial cell junctions. This study suggests a mechanism by which ongoing low level exposures perpetuate airway inflammation after an inducing toxic inhalation. A possible overlap between reactive airways dysfunction syndrome, reactive upper-airway dysfunction syndrome and the multiple chemical sensitivity syndrome is suggested.

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions.

Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin are structurally related peptides with N-terminal 6-7 amino acid ring structures linked by a disulfide bridge and with amidated C-termini. Among the related bioactive peptides, the structures of the calcitonin receptor and subtypes thereof have been identified so far through molecular cloning. Cross-reaction between receptors of calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin, as well as overlapping biological actions, anticipate that the respective receptors belong to a family of G-protein-coupled receptors that include those of parathyroid hormone, secretin and vasointestinal peptide.

Neurotrophins and the neuroendocrine brain: different neurotrophins sustain anatomically and functionally segregated subsets of hypothalamic dopaminergic neurons

Hypothalamic neurons control a variety of important hormonal and behavioral functions. Little is known, however, about the neurotrophic factors that these neurons may require for survival and/or maintenance of their differentiated functions. We conducted experiments to examine this issue, utilizing a combination of immunohistochemical, in situ hybridization and cell culture approaches. We found that the low affinity receptor for nerve growth factor (p75 NGFR) is present in small subsets of hypothalamic peptidergic neurons identified as such by their content of galanin, luteinizing hormone-releasing hormone (LHRH) and vasointestinal peptide (VIP). More prominently, however, examination of hypothalamic dopaminergic (DA) neurons for the presence of p75 NGFR-like immunoreactivity revealed that the receptor was present on tyrosine hydroxylase (TH)-positive neurons of the zona incerta and periventricular region, but not on neuroendocrine DA neurons of the tuberoinfundibular region. In situ hybridization experiments using a p75 NGFR cRNA confirmed this distribution. Regardless of the presence or absence of p75 NGFR, neither DA group expresses trkA mRNA, indicating that these two major hypothalamic subsets of DNA neurons are NGF-insensitive. A substantial fraction of TH mRNA-positive cells in the zona incerta expresses trkB mRNA, which encodes the receptor for brain derived neurotrophic factor (BDNF); in turn BDNF supports the in vitro survival of hypothalamic TH neurons bearing p75-NGFR, suggesting that BDNF is trophic for DNA neurons of the zona incerta. In contrast, tuberoinfundibular DA neurons do not express trkB mRNA, but some have trkC mRNA, which encodes the receptor for neurotrophin-3 (NT-3). The in vitro survival of TH neurons devoid of p75-NGFR is supported by NT-3, implying that NT-3 may be trophic for a subset of tuberoinfundibular DA neurons. These results suggest that, in spite of expressing an identical neurotransmitter phenotype, anatomically and functionally segregated DA neurons of the neurodendocrine brain are sustained by different neurotrophic factors.

A Human Orphan Calcitonin Receptor-like Structure

A novel calcitonin receptor-like protein of 461 amino acids with seven putative transmembrane domains has been identified through molecular cloning in a cDNA library of the human cerebellum. 91% and 56% of the amino acids are identical in a rat orphan calcitonin receptor-like sequence and the human calcitonin receptor, respectively. 5.2 kb mRNA is predominantly expressed in the lung, heart and kidney. Specific binding of 125I -labeled salmon calcitonin and human calcitonin gene-related peptide-I to COS-7 cells transiently transfected with the receptor cDNA was less then 0.5%. Cellular cAMP accumulation was indistinguishable in cDNA transfected and non-transfected control COS-7 and renal tubular cells from the American opossum stimulated with human and salmon calcitonin, human calcitonin gene-related peptide-I and -II, human amylin, human adrenomedullin, lizard helodermin, salmon stanniocalcin and chicken parathyroid hormone-related protein. The receptor-like protein whose ligand remains to be discovered belongs to the family of receptors of calcitonin, parathyroid hormone, secretin, vasointestinal peptide and pituitary adenylate cyclase-activating polypeptide.

Development and validation of a homologous radioimmunoassay using a biologically active recombinant turkey prolactin

1. A new homologous radioimmunoassay has been developed for the measurement of turkey prolactin. 2. A 25000 kDa purified recombinant derived turkey prolactin (rtPRL), the biological activity of which was tested using a crop sac assay, was used as immunogen for the production of rabbit antiserum. In this biological test, the rtPRL was as active as the ovinePRL. 3. The radioligand (rtPRL) was labelled with 125I and the assay allowed the detection of standard doses of rtPRL ranging from 400 pg/tube to 50 ng/tube. 4. No cross reaction with chicken luteinising hormone and recombinant chicken growth hormone was detected. 5. The within and between assay coefficients of variability were 5.0 +/- 2.7% and 16.3%, respectively. The overall mean recovery ratio was 1.01. 6. The dose-response curves obtained with serial dilution of plasma and pituitary from turkey hens at different physiological stages and from male turkeys were parallel to those obtained with standard rtPRL. 7. The measured concentration of prolactin was 5 times higher in plasma from incubating than laying turkey hens, and the pituitaries from incubating hens contained 2 and 4 times more prolactin than those of laying and out of lay hens or males, respectively. 8. To further assess the validity of the assay, we measured changes in plasma concentration of prolactin in turkeys following stimulation with chicken vasointestinal peptide (cVIP). A single injection of 1 or 10 micrograms/kg body weight of cVIP to laying hens produced a large and rapid increase in plasma prolactin. 9. This new radioimmunoassay appears to be high for the measurement of turkey prolactin.

A combined evaluation of biochemical and morphological changes during human neuroblastoma cell differentiation.

1. The effects of retinoic acid, gamma-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction. 2. Cell morphology was strongly affected by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and gamma-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects. 3. Acetylcholinesterase activity was significantly stimulated by retinoic acid and by gamma-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold. 4. The cellular catecholamine and related metabolite content was lowered by retinoic acid and gamma-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation. 5. Finally, the cell-associated vasointestinal peptide level was strikingly increased by gamma-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. 6. It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.

Nonadrenergic, noncholinergic airway inhibitory nerves

Nonadrenergic, noncholinergic (NANC) nerves, which cause relaxation of airway smooth muscle, have been described in several species including man. Stimulation of efferent vagus nerves during cholinergic and adrenergic blockade induces a pronounced bronchodilation in the cat. In more recent studies in man, capsaicin inhalation or mechanical irritation of the larynx, under conditions of cholinergic and adrenergic blockade, have been shown to cause a transient bronchodilator response. There is some evidence that neuropeptides such as vasointestinal peptide (VIP) or peptide histidine methionine (PHM) may be the neurotransmitter of NANC nerves, but this is not conclusive. Nitric oxide may be another neurotransmitter. In mild asthma, the NANC bronchodilator response is similar to that observed in normal subjects; on the other hand, a reduction in VIP immunoreactivity has been reported in more severe patients. The contribution of NANC dilator nerves in pathophysiological situations is not known, but their effect may be modulated during allergic responses. Use of antagonists or inhibitors of putative neurotransmitters, and molecular biological techniques will be useful in defining both the physiological and pathophysiological roles of NANC inhibitory nerves in the airways.

Growth hormone releasing hormone receptors on thymocytes and splenocytes from rats

In the present study, we determined that rat mononuclear leukocytes possess specific receptors for growth hormone releasing hormone (GHRH). The results show that the binding of 125I-labeled GHRH to spleen and thymic cells was saturable and of a high affinity, approximately 3.5 and 2.5 n M for thymus and spleen cells, respectively. The Scatchard analysis revealed a binding capacity of approximately 54 and 35 fmol per 10 6 cells on thymus and spleen, respectively. The binding of GHRH was not competed by 10 −6 M growth hormone, corticotropin releasing factor, substance P or luteinizing hormone releasing hormone and vasointestinal peptide (VIP). Partial characterization of the receptor was accomplished by crosslinking 125I-labeled GHRH to thymus cells with disuccinimidyl suberate and polyacrylamide gel electrophoresis. Autoradiography of dried gels showed two major components in leukocytes and pituitary cells at approximately 42 and 27 kDa which could be diminished by unlabeled GHRH. The treatment of leukocytes with GHRH (10 n M) rapidly increased the intracellular free calcium concentration from a basal level of 70 ± 20 n M to a plateau value of 150 ± 20 n M in 6 min after stimulation. The functional activity of GHRH receptors was studied further by measuring lymphocyte proliferative responses and the increase in the level of cytoplasmic GH RNA. The presence of GHRH alone resulted in a dose-dependent increase in thymidine and uridine incorporation and a dose-dependent increase in the levels of GH RNA in the cytoplasm. Taken together, the results show that lymphocytes contain specific receptors for GHRH that are coupled to important biological responses and further support the concept of bidirectional communication between the immune and neuroendocrine tissues.

Expression of luteinising hormone-β subunit chloramphenicol acetyltransferase (LH-β-CAT) fusion gene in rat pituitary cells: Induction by cyclic 3′-adenosine monophosphate (cAMP)

In this study we determined the activity of the rat luteinising hormone-β gene promoter in a heterologous rat pituitary cell line (GH 3 cells). 1.7 kb of LH-β 5′ flanking sequence and the first 5 bp of the 5′ untranslated region were ligated to the chloramphenicol acetyltransferase (CAT) receptor gene (LH-β-CAT) and transiently transfected by calcium phosphate precipitation into subconfluent cultures of GH 3 cells. Basal low-level CAT activity was only detected in GH 3 cells, being absent in two non-pituitary cell lines (BeWo and HeLa) RNase analysis revealed that mRNA from transfected GH 3 cells protected a fragment of labelled antisense probe of correct size for transcription initiation from the LH-β CAP site, confirming that promoter activity reflected correctly initiated LH-β-CAT fusion gene transcripts. CAT activity was consistently induced by an average of 3–5-fold from the full-length 1.7 kb promoter, in a dose- and time-dependent manner, by forskolin, dibutyryl cAMP, and 8-bromo cAMP implying presence of a cAMP-responsive cis-acting domain in the LH-β promoter region. Transfection of deletion mutants Δ-615-CAT, Δ-385-CAT and Δ-250-CAT each reduced forskolin inducibility to 1.7-fold but did not abolish induction completely suggesting a domain between −1.7 and −0.6 kb contained a cAMP-responsive element(s) (CRE). Further deletion of LH-β 5′ flanking sequences to Δ-85-CAT restored forskolin induction to wild-type levels (3–5-fold), suggesting the presence of a weak inhibitory element between −600 and −85 kb, and a cAMP-responsive domain in the proximal promoter region. The LH-β promoter does not contain perfect tandem repeat palindromic CRE DNA sequences, though there are several octanucleotide sequences differing by only 1 bp from AP-2 binding sites, the consensus CRE, and the vasointestinal peptide gene CRE. Although these data suggest that the LH-β gene is cAMP responsive this is likely mediated by several and complex protein interactions with multiple DNA sequences in the proximal and distal LH-β promoter enhancer.