Intestinal vasoactive peptide receptors in enterocytes : specific binding and stimulation of cyclic AMP]

Receptors for the vasoactive intestinal peptide (VIP) were characterized on enterocytes isolated from Rat small intestine. Native VIP inhibited competitively the binding of 125I-VIP to enterocytes and strongly stimulated cyclic AMP production; both these effects were observed for concentrations of VIP as low as 0.5-1.0 ng/ml (0.15-0.30 nM) which are compatible with the VIP concentration in the gut.     

Priming effect of vasoactive intestinal peptide on the respiratory burst of neutrophils non-mediated by plasma membrane receptors

Vasoactive intestinal peptide (VIP) primed the respiratory burst of human neutrophils in response to phorbol myristate acetate. Maximal and half-maximal effects were achieved at 10 and 0.5 nM VIP respectively. The absence of plasma membrane receptors to VIP in neutrophils suggests that priming of the respiratory burst should be considered as a side effect of VIP. However, from the above indicated concentration range, the priming of the neutrophil by VIP cannot be considered as a pharmacological effect. The enhancement of the formation of reactive oxygen metabolites by VIP may be important in the pathology of VIP-producing tissues.     

Combined effects of testosterone and estradiol on the ventral lobe of the rat postate in organ culture]

After organ culture without hormone, the epithelial gland cells of Rat veantral prostate undergo atrophic changes, whereas the interstitial stroma components tend to increase. Estradiol (1-1,000 nM),added to the culture medium, is ineffective. On the contrary, testosterone (1-100 nM) maintains epithelial cells and prevents the increase of interstitial stroma. When estradiol (1-1,000 nM) is combined with a physiological concentration of testosterone (1-4 nM), the epithelial cells are well maintained, but the inhibitory action of testosterone on the stroma is counteracted so that the glandular epithelium and the interstitial stroma are both stimulated. However, when testosterone is used at supraphysiological (10-100 nM) concentrations, estradiol is completely ineffective and the structure of the prostate is identical to the one given by the androgen alone.     

Binding of estradiol to synaptosomal mitochondria: physiological significance

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.     

Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder

Electrical changes and direct effects on Na-K ATPase activity induced by an endogenous digitalis-like natriuretic factor (NF), NH4Cl and ouabain were studied in toad bladders. NF inhibited the SCC and the Na-K ATPase activity in a similar manner to ouabain, but induced a greater increase in calculated direct current resistance (R) (p less than 0.05). NH4Cl was a weak inhibitor of Na-K ATPase activity, although it produced steeper SCC inhibition slopes than those observed with ouabain or NF (p less than 0.01). The data suggested the same mechanism of action of NF and ouabain on the sodium pump, with an additional effect of the former on apical sodium permeability of the cells and/or closure of paracellular routes leading to an increased tissue resistance. In contrast, the effects of NH4Cl were mostly compatible with intracellular inhibition of apical sodium entry into the cell.     

Secretion of insulin and of glucagon by the perfused pancreas of newborn rats: effect of vasoactive intestinal peptide Vip]

A method is described for perifusion of the splenic part of the pancreas from 48-64 hour-old rat. In different basal conditions, the secretion of insulin and glucagon is stable and reproducible for 90 mn. The addition of the vasoactive intestinal peptide (VIP) to these perifusion media, at a concentration as low as 2 ng/ml, determines a remarkable increase of insulin and of glucagon secretion. These results suggest the possibility of a VIP action in the physiology of endocrine pancreas.     

Lithium and phorbol ester modify the activating capacity of ascidian spermatozoa

In this paper we have shown, using the whole-cell voltage clamp technique, that two parameters of the fertilization current in ascidian eggs may be modified by exposing spermatozoa to lithium or to phorbol ester. When spermatozoa were pre-treated in 250 mM lithium sea water for up to 30 min there was a significant increase in the mean initial slope of the fertilization current, from 116±90 to 169±84 pA/s (p<0.05). The peak current increased from 1371±1079 to 1719±1052 pA (p>0.05). Pre-treatment in 200–600 nM phorbol 12-myristate 13-acetate also increased the activating capacity of ascidian sperm, as monitored by a significant increase in the initial slope current in control eggs; however, there was no increase in peak current. Furthermore, we have shown, using NH₄Cl, that an increase in intracellular pH alone is insufficient to change the activating capacity of spermatozoa. This is the first report showing that the kinetics of an egg activation event depend upon the physiological status of the spermatozoon.     

A study of vasoactive intestinal polypeptide (VIP) stimulated intestinal fluid secretion in rat and its inhibition by indomethacin

Summary 4 groups of rats were studied under anaesthesia to assess the effect of VIP and the influence of the prostaglandin synthetase inhibitor indomethacin in isolated bowel loops. VIP produced a highly significant increase in the luminal fluid content and this was completely inhibited by addition of indomethacin.     

A study of vasoactive intestinal polypeptide (VIP) stimulated intestinal fluid secretion in rat and its inhibition by indomethacin

4 groups of rats were studied under anaesthesia to assess the effect of VIP and the influence of the prostaglandin synthetase inhibitor indomethacin in isolated bowel loops. VIP produced a highly significant increase in the luminal fluid content and this was completely inhibited by addition of indomethacin.     

Effect of halogenmethylenebisphosphonates on bone cells in culture and on bone resorption in vivo

Dihalogenmethylenebisphosphonates increase alkaline phosphatase activity and fatty acid oxidation in calvaria cells in culture (Cl2MBP greater than Br2MBP approximately equal to F2MBP). The monohalogen ClMBP and the non-halogenated analogues are less active on phosphatase and inactive on or inhibitory towards fatty acid oxidation. The three dihalogenbisphosphonates and ClMBP inhibit bone resorption in vivo, Cl2MBP most strongly.     

K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

Vasoactive intestinal peptide (VIP) occurs in nerves of the pineal gland

Summary Nerves staining with antibodies against vasoactive intestinal peptide (VIP) were detected in the pineal gland of the rabbit, cat and pig. VIP nerves were numerous in the cat but few in the rabbit and pig. A particularly rich VIP nerve supply was noted in the pineal stalk of the cat. The nerves were predominantly located around small blood vessels. Occasionally, nerve fibres were seen in the glandular parenchyma without obvious relation to blood vessels.Grant support from the Swedish Medical Research Council (04X-4499).     

Differential stimulation of signaling pathways initiated by Edg-2 in response to lysophosphatidic acid or sphingosine-1-phosphate

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are produced during cell activation and have multiple effects on cells. A family of seven transmembrane-spanning domain G-protein-coupled receptors, named Edg, mediate these effects of LPA and S1P. In this study, transient overexpression of Edg-2 sensitized MG63 human osteosarcoma cells to both LPA- and S1P-mediated stimulation of fibronectin matrix deposition and actin stress fiber formation. Both lipids were active in the 1-20 nM concentration range on cells transfected with Edg-2 as compared to the 10-200 nM range on mock-transfected cells. The signaling pathway for matrix deposition by Edg-2-transfected cells was Rho dependent. Overexpression of Edg-2 also caused a tenfold decrease in the concentration of either LPA or S1P that activated MAPKinase (Erkl/2) in MG63 cells. LPA- or S1P-stimulated activation of Erkl/2 was Gi dependent. These results indicate that, in MG63 cells, Edg-2 mediates actin stress fiber formation, fibronectin matrix assembly, and MAPKinase activation in response to either LPA or S1P.     

Effect of thiamine hydrochloride on the blood level of 2-formyl 1-methyl pyridinium oxime chloride (2-PAM.Cl) in rats

Summary The biological half-life of 2-PAM.Cl was found to increase in female rats pretreated with thiamine hydrochloride (10 mg/kg i.m.). No such effect was observed in the male rats.Dr B.L. Chowdhri very kindly synthesized the compound 2-PAM.Cl in the Department of Chemistry of this establishment for this work.The authors are thankful to Dr P.K. Ramachandran, Director, and Dr A.K. Chatterjee, Deputy Director, Defence Research and Development Establishment, Gwalior, for their keen interest in this work.     

Action of histamine and vasoactive intestinal peptide (VIP) on cyclic AMP in gastric glands isolated from human fetal stomach

Histamine and VIP produce an elevation of cAMP production in gastric glands isolated from the human fetal stomach at 15 weeks gestation. These effects were attributed to the activation of 2 distinct receptor-cAMP systems, one being sensitive to histamine in parietal cells, and the other being sensitive to VIP in muco-peptic cell populations. The results suggest that histamine and VIP may play a role in inducing gastric secretion during fetal life in man.     

Action of histamine and vasoactive intestinal peptide (VIP), on cyclic AMP in gastric glands isolated from human fetal stomach

Summary Histamine and VIP produce an elevation of cAMP production in gastric glands isolated from the human fetal stomach at 15 weeks of gestation. These effects were attributed to the activation of 2 distinct receptor-cAMP systems, one being sensitive to histamine in parietal cells, and the other being sensitive to VIP in muco-peptic cell populations. The results suggest that histamine and VIP may play a role in inducing gastric secretion during fetal life in man.     

The regulatory peptide system of the large bowel in equine grass sickness

Summary In recent years, distinct changes in regulatory peptides have been found in a number of gastrointestinal diseases. Grass sickness is a fatal disease of horses for which the etiology has yet to be fully ascertained. In this study, the peptide-containing nerves and ganglionic and mucosal endocrine cells of the ileum, colon and rectum were investigated in horses with sub-acute or chronic grass sickness and compared with normal controls using immunocytochemistry, at both the light and electron microscopical levels, and radioimmunoassay. A substantial loss of both peptide-containing cells and nerves was found in all of the sick horses, particularly in the ileum. Electron microscopy revealed marked degeneration of nerves in the gut wall. fibers containing granules immunostained for substance P or VIP, using the immunogold staining technique, underwent extensive degranulation in grass sickness, with the formation of multiple vacuoles.Radioimmunoassay of peptide content also showed that the most drastic changes occurred in the ileum. For example, VIP content was significantly reduced from 109±19.8 (mean±SEM) pmoles/g in controls to 6.8±1.4 pmoles/g in grass sickness (p<0.001) and substance P from 65.9±8.1 to 31.3±9.5 (p<0.02). These results may have applications in the diagnosis and treatment of grass sickness.The authors gratefully acknowledge the financial support of the Wellcome Trust and the Grass Sickness Fund.     

Opioids and cytosolic calcium in rat anterior pituitary: dynorphin preparation showed LHRH-like action due to contamination

The effect of dynorphin A-(1-13) (Dyn A-(1-13] and other opioids on the cytosolic free calcium concentration [(Ca2+]i) in rat anterior pituitary cells was examined using the fluorescent indicator fura-2. A commercial synthetic Dyn A-(1-13) preparation elevated [Ca2+]i. Results, which were obtained with receptor antagonists, and in LHRH receptor radioligand binding studies as well as by HPLC combined with LHRH radioimmunoassay, strongly suggest that this effect of the dynorphin preparation was due to contamination with a LHRH-like compound. Dyn A-(1-13), purified by HPLC, as well as Dyn A-(2-13), [Leu5]enkephalin, beta-endorphin, morphine, or U50,488H had no effect on [Ca2+]i. LHRH caused a rapid increase in [Ca2+]i by about 50 nM which was blocked by the LHRH antagonist, [D-pGlu1,D-Phe2,D-Trp3,6] LHRH.     

A glucagon-secretin-like peptide stimulates the intrinsic nervous plexus of guinea pig gallbladder

The role of vasoactive intestinal peptide (VIP), as a possible neurotransmitter of the intrinsic nerve plexus in the guinea pig gallbladder, was investigated by monitoring spontaneous contractile activity. VIP receptor antagonist (4 Cl-D-Phe6, Leu 17)-VIP did not produce any effect on muscular tone and spontaneous activity, whereas (N-Ac-Tyr1, D-Phe2)-GRF-(1-29)-NH2, (14-GRF analog), which is known to stimulate digestive enzyme secretion by interacting with the VIP-preferring receptors, greatly increased the amplitude and frequency of waves as well as the muscular tone. Since VIP receptor antagonist acts selectively as a competitive antagonist for the action of VIP, we conclude that the gallbladder inhibitory intrinsic plexus neurotransmitter is not VIP, but a member of the glucagon-secretin family of peptides.     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A2 and C strongly (ED50 congruent to 4 X 10(-2) and 4 X 10(-1) micrograms/ml, respectively) and rapidly reduced 125I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.