Histamine protects against NMDA-induced necrosis in cultured cortical neurons through H receptor/cyclic AMP/protein kinase A and H receptor/GABA release pathways

Using histamine and the H3 receptor antagonist thioperamide, the roles of histamine receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Within 3 h of intense NMDA insult, most neurons died by necrosis. Histamine reversed the neurotoxicity in a concentration-dependent manner and showed peak protection at a concentration of 10(-7) m. This protection was antagonized by the H2 receptor antagonists cimetidine and zolantidine but not by the H1 receptor antagonists pyrilamine and diphenhydramine. In addition, the selective H2 receptor agonist amthamine mimicked the protection by histamine. This action was prevented by cimetidine but not by pyrilamine. 8-Bromo-cAMP also mimicked the effect of histamine. In contrast, both the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine and the cAMP-dependent protein kinase inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide reversed the protection by histamine. Thioperamide also attenuated NMDA-induced excitotoxicity, which was reversed by the H3 receptor agonist (R)-alpha-methylhistamine but not by pyrilamine and cimetidine. In addition, the protection by thioperamide was inhibited by the GABA(A) receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by thioperamide was due to increased GABA release in NMDA-stimulated samples. These results indicate that not only the H2 receptor/cAMP/cAMP-dependent protein kinase pathway but also the H3 receptor/GABA release pathway can attenuate NMDA-induced neurotoxicity.     

Necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and cholecystokinin2 receptors on parietal cells in isolated mouse stomach

The existence of a direct action of acetylcholine and gastrin on muscarinic M3 and cholecystokinin2 (CCK2) receptors on gastric parietal cells has not yet been convincingly established because these stimulated acid secretions are remarkably inhibited by histamine H2 receptor antagonists. In the present study, we investigated the necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and CCK2 receptors on parietal cells using an isolated mouse stomach preparation. Bethanechol (10-300 microM) produced a marked increase in acid output and this increase was completely blocked by famotidine (10 microM). In the presence of famotidine, bethanechol (1-30 microM) augmented the acid secretory response to dibutyryl AMP (200 microM) in a concentration-dependent manner. The augmentation was blocked by atropine (1 microM), 4-DAMP (0.1 microM), a muscarinic M3-selective antagonist, and by Ca2+ exclusion from the serosal nutrient solution. Pentagastrin (0.3-3 microM) also concentration-dependently stimulated gastric acid secretion, but the effect was completely inhibited by famotidine. In the presence of famotidine, pentagastrin (0.1-0.3 microM) elicited a definite potentiation of the acid secretory response to dibutyryl cyclic AMP (200 microM). This potentiation was inhibited by YM022 (1 microM), a CCK2 receptor antagonist, and by exclusion of Ca2+ from the serosal nutrient solution. The present results suggest that gastric acid secretion via the activation of muscarinic M3 and CCK2 receptors on the parietal cells is induced by activation of the cyclic AMP-dependent secretory pathway.     

A calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells

The role of calmodulin in the regulation of histamine-stimulated parietal cell function was studied in isolated rat parietal cells using [14C]aminopyrine uptake as a quantitative index of acid production. In enriched (77-87%) intact parietal cells the calmodulin antagonist naphthalene sulfonamide W 7 dose-dependently inhibited the response to 10(-4) M histamine (IC50: 2 X 10(-6) M). The mechanism of this inhibition was examined further with two other stimuli of H+-production: forskolin which directly activates the parietal cell adenylate cyclase without interacting at the histamine H2-receptor and dbcAMP which mimics the biological action of cAMP without preceding activation of adenylate cyclase. W 7 effectively inhibited the responses to 10(-4) M forskolin (IC50: 6 X 10(-7) M), 10(-3) M dbcAMP (IC50: 10(-6) M) and to 10(-2) M K+ (IC50: 3 X 10(-6) M). The action of W 7 followed non-competitive kinetics since the antagonist reduced the entire range of the concentration-response curves without shifting them rightwards towards higher concentrations of the respective stimulants. The effect of W 7 was reversed by washing the cells. ATP-induced [14C]aminopyrine uptake into digitonin-permeabilized oligomycin-inhibited parietal cells reflects H+-production independent of oxidative phosphorylation and was also inhibited by W 7 (IC50: 10(-5) M). Inhibition of K+-stimulated H+/K+-ATPase activity required even higher W 7-concentrations (IC50: 1.4 X 10(-4) M). Our data suggest that calmodulin might be involved in the intracellular mediation of the response to histamine. Between histamine-induced cAMP-generation and the H+-secreting tubulovesicular system W 7 seems to inhibit an intracellular step that finally activates the H+/K+-ATPase. Yet, direct inhibition of the ATPase requires W 7 concentrations of questionable specificity and is unlikely to be the mechanism behind the action of W 7 on the parietal cell response to histamine.     

Naturally occurring opioid peptides modulate H+-production by isolated rat parietal cells

The rationale for the present study was to determine the effects of naturally occurring opioid peptides on H+-production by isolated rat parietal cells as indirectly measured by [14C]-aminopyrine uptake. In crude preparations (18 to 25% parietal cells) and in enriched (80 to 90%) parietal cell fractions stimulation by submaximal histamine- or dibutyryl cAMP-concentrations (10(-6)-10(-4) mol/l) was augmented by 20-30% in the presence of methionine-enkephalin (Met-Enk) and Met-Enk Arg6Phe7 (10(-7) to 10(-5) mol/l). This augmentation was blocked by the opiate receptor antagonist (-)naloxone (10(-6) mol/l) suggesting specificity of the action of Met-Enk and Met-Enk Arg6Phe7. At 10(-6) mol/l (-)naloxone did not exert nonspecific toxic effects. Yet, even in the absence of exogenous opioids, histamine-induced H+-production was inhibited by 3 X 10(-5) or 10(-4) mol/l (-)naloxone. Since similar inhibition occurred with (+)naloxone, an inactive stereoisomer which does not interact with opiate receptors, effects of (-)naloxone at concentrations above 10(-5) mol/l must be considered nonspecific. We conclude that Met-Enk and Met-Enk Arg6Phe7 have no effect on basal, but augment stimulated H+-production by a direct effect on the parietal cells. At nontoxic concentrations (-)naloxone antagonizes this augmentation indicating that it is mediated by specific opiate receptors on the parietal cells.     

Histamine is a potent inducer of IL-18 and IFN-gamma in human peripheral blood mononuclear cells

Histamine (10-7 to 10-4 M) concentration-dependently stimulated the production of IL-18 and IFN-gamma and inhibited the production of IL-2 and IL-10 in human PBMCs. Histamine in the same concentration range did not induce the production of IL-12 at all. The stimulatory or inhibitory effects of histamine on cytokine production were all antagonized by H2 receptor antagonists ranitidine and famotidine in a concentration-dependent manner, but not by H1 and H3 receptor antagonists. Selective H2 receptor agonists, 4-methylhistamine and dimaprit, mimicked the effects of histamine on five kinds of cytokine production. The EC50 values of histamine, 4-methylhistamine, and dimaprit for the production of IL-18 were 1.5, 1.0, and 3.8 microM, respectively. These findings indicated that histamine caused cytokine responses through the stimulation of H2 receptors. All effects of histamine on cytokine responses were also abolished by the presence of either anti-IL-18 Ab or IL-1beta-converting enzyme/caspase-1 inhibitor, indicating that the histamine action is dependent on mature IL-18 secretion and that IL-18 production is located upstream of the cytokine cascade activated by histamine. The addition of recombinant human IL-18 to the culture concentration-dependently stimulated IL-12 and IFN-gamma production and inhibited the IL-2 and IL-10 production. IFN-gamma production induced by IL-18 was inhibited by anti-IL-12 Ab, showing the marked contrast of the effect of histamine. Thus histamine is a very important modulator of Th1 cytokine production in PBMCs and is quite unique in triggering IL-18-initiating cytokine cascade without inducing IL-12 production.     

Localization of (3H)histamine and (3H)prostaglandin E2 receptors in isolated cells of rat gastric mucosa

Isolated cells of rat gastric mucosa were obtained by treatment of rat stomach with pronase. Two fractions were isolated, one of which was rich (up to 90%) and the second one poor (to 25%) of parietal cells. Using specific antagonists and agonists of H1- and H2-receptors of histamine (diphenhydramine, metiamide, cimetidine, impromidine, dimaprit) the H2-receptors of histamine were shown to be localized in parietal cells. A preferential binding of (3H)prostaglandin E2 by the receptor proteins of plasma membranes of non-parietal (presumably mucoid) cells was found. The data obtained indicate that rat gastric mucosa contains receptors of histamine and PGE2 which differ in their intracellular localization and strictly selectively bind (3H)histamine and (3H)PGE2. It is assumed that the starting point in the mechanism of action of these intercellular regulators on gastric secretion is probably the process of their specific recognition by the protein receptors localized in functionally different cells.     

The adenylate cyclase system and calcitonin secretion from perfused dog thyroid lobes

The aim of the study was to assess the involvement of the adenylate cyclase system in calcitonin (CT) secretion from thyroidal C-cells. The cAMP analogues Br-cAMP (10(-6) and 10(-4) mol/l) and DB-cAMP (10(-4) mol/l) and the activators of adenylate cyclase cholera toxin (0.1 microgram/ml and 5 micrograms/ml) and forskolin (10(-7) mol/l and 10(-5) mol/l) were infused for 6 min periods in perfused dog thyroid lobes. CT was measured in thyroid effluent by radioimmunoassay. Br-cAMP and cholera toxin did not alter basal CT secretion. DB-cAMP had a minimal stimulatory effect and forskolin 10(-5) mol/l a moderate stimulatory effect. This was much less than the effect of increasing perfusate Ca++ from 1.5 to 2.0 mmol/l. 10(-4) mol/l Br-cAMP increased the response to Ca++ with approximately 50 per cent. These results suggest that the activity of the adenylate cyclase system of the C-cells by itself is of little importance for CT secretion, but that it may have a role as modulator of the response to Ca++.     

Modelling of H2 antagonists and tridimensional modelling of H2 receptor interactions. Part 2

The interactions between the H2 antagonists cimetidine, ranitidine and famotidine with a basic molecular model for the histamine H2 receptor have been analyzed. The calculated potential energies of the antagonist-H2 receptor complexes follow an order consistent with the published binding data, indicating that famotidine is the best H2 receptor ligand. Comparison with the interactions found for histamine and this H2 receptor model suggests that the protonated imiddazole moiety of cimetidine, the dimethylammonio moiety of protonated ranitidine and the protonated guanidinyl moiety of famotidine are bioisosteric with the protonated aliphatic amine group of histamine. Asp 98 in helix 3 appears to be the main residue for antagonist recognition, but some residues in helix 5 may be involved, apparently by serving to guide the antagonist into the binding pocket.     

Receptor binding of pancreatic spasmolytic polypeptide (PSP) in rat intestinal mucosal cell membranes inhibits the adenylate cyclase activity

The recently isolated pancreatic spasmolytic polypeptide, PSP, interacted with specific binding sites in the gastrointestinal tract and inhibited the adenylate cyclase activity in rat intestinal mucosal cell membranes. The binding sites appeared to be heterogeneous and Scatchard analysis of the binding data indicated the presence of at least two classes of sites. The high-affinity low-capacity binding sites and the low-affinity high-capacity binding sites had apparent dissociation constants of 1.3 X 10(-7) mol/l and 4.2 X 10(-6) mol/l, respectively. The PSP induced inhibition of the adenylate cyclase activity was independent of the stimulatory state of the enzyme. The basal activity as well as that stimulated by VIP and secretin was half maximally inhibited at approximately 3 X 10(-5) mol/l of PSP. The inhibitory effect of PSP was independent of the agonist concentration employed. PSP did not affect the receptor binding of VIP nor did VIP affect the receptor binding of PSP.     

Cellular origin and release of intrinsic factor from isolated rat gastric mucosal cells

The cellular content and secretion of intrinsic factor was measured by [57Co]cyanocobalamin binding using isolated rat gastric mucosal cells. The intrinsic factor/R-protein ratio was above 9:1 as evaluated by specific anti-intrinsic factor antibodies. In unfractionized cells with 23 +/- 1.3% parietal cells the intrinsic factor content of 148 +/- 47 fmol/10(6) cells remained almost unchanged over 3 h, whereas basal secretion rose up to 57 +/- 10. In fractionized cells (Percoll) with 3-85% parietal cells most intrinsic factor was found in the parietal cell-depleted fraction (content: 441 +/- 30, secretion/3 h: 139 +/- 16, mean formation/h: 50 +/- 12 fmol/10(6) cells). The intrinsic factor content of the different cell fractions correlated with that of pepsin. [14C]Aminopyrine uptake, an indirect measure of parietal cell H+ production, was inversely related. Carbachol (1 X 10(-6)-10(-3) mol/l) stimulated intrinsic factor secretion, 1 X 10(-3) mol/l being maximally effective (90 +/- 8% above basal). This response was inhibited by atropine and pirenzepine, but not by prostaglandin E2 (PGE2) and somatostatin. Dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP, 43 +/- 7%) and hexoprenaline (24 +/- 5%) enhanced intrinsic factor secretion less effectively and pentagastrin like histamine lacked any stimulatory effect. We conclude that in the rat intrinsic factor is produced and released from chief cells mainly under cholinergic control.     

Synthesis and pharmacological activity of fluorescent histamine H2 receptor antagonists related to potentidine

Fluorescently labeled histamine H(2) receptor antagonists were synthesized starting from N-cyano-N'-[3-(3-piperidin-1-ylmethylphenoxy)propyl]guanidines with an additional N"-omega-aminoalkyl substituent (chain lengths 2-8 methylene groups) or from 3-(3-piperidin-1-ylmethylphenoxy)propylamine. The primary amino group was derivatized with various fluorophores (fluorescein, acridine, dansyl, nitrobenzoxadiazole (NBD), indolo[2,3-a]quinolizine). On the isolated spontaneously beating guinea pig right atrium most of the fluorescent probes were only weakly active, however, the NBD-labeled substances proved to be potent histamine H(2) receptor antagonists achieving pA(2) values in the range of 7.5-8.0, comparable to the activity of famotidine.     

Histamine interaction on surface recognition sites of H2-type in parietal and non-parietal cells isolated from the guinea pig stomach

In gastric cells isolated by pronase digestion from the guinea pig, histamine stimulated cAMP production in 3 fundic cell fractions (EC50 = 1.6--2 x 10(-4) M) enriched in parietal (94%), peptic (63%) and mucous cells (87%) as well as in antral cells (EC50 = 4 x 10(-4) M) that are devoid of parietal cells. Histamine stimulations were completely inhibited by the H2 antagonist cimetidine (Ki = 0.27--0.57 x 10(-6) M) or by the H1 antagonist diphenhydramine, but at 100-times lower potency (Ki = 22--45.7 x 10(-6) M), indicating the presence of histamine H2 receptors in parietal and nonparietal cells of the guinea pig gastric mucosa.     

B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.     

Stimulatory effects of endogenous and exogenous nitric oxide on gastric acid secretion in anesthetized rats

We previously reported the stimulatory effect of endogenous nitric oxide (NO) on gastric acid secretion in the isolated mouse whole stomach and histamine release from gastric histamine-containing cells. In the present study, we investigated the effects of endogenous and exogenous NO on gastric acid secretion in urethane-anesthetized rats. Acid secretion was studied in gastric-cannulated rats stimulated with several secretagogues under urethane anesthesia. The acid secretory response to the muscarinic receptor agonist bethanechol (2 mg/kg, s.c.), the cholecystokinin(2) receptor agonist pentagastrin (20 microg/kg, s.c.) or the centrally acting secretagogue 2-deoxy-D-glucose (200 mg/kg, i.v.) was dose-dependently inhibited by the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 or 50 mg/kg, i.v.). This inhibitory effect of L-NNA was reversed by a substrate of NO synthase, L-arginine (200 mg/kg, i.v.), but not by D-arginine. The histamine H(2) receptor antagonist famotidine (1 mg/kg, i.v.) completely inhibited the acid secretory response to bethanechol, pentagastrin or 2-deoxy-D-glucose, showing that all of these secretagogues induced gastric acid secretion mainly through histamine release from gastric enterochromaffin-like cells (ECL cells). On the other hand, histamine (10 mg/kg, s.c.)-induced gastric acid secretion was not inhibited by pretreatment with L-NNA. The NO donor sodium nitroprusside (0.3-3 mg/kg, i.v.) also dose-dependently induced an increase in acid secretion. The sodium nitroprusside-induced gastric acid secretion was significantly inhibited by famotidine or by the soluble guanylate cyclase inhibitor methylene blue (50 mg/kg, i.v.). These results suggest that NO is involved in the gastric acid secretion mediated by histamine release from gastric ECL cells.     

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

Both endothelium and afferent nerve endings play a role in acetylcholine-induced renal vasodilation

We investigated the nature and signaling pathways of endothelium- and sensory-nerve ending-derived substances involved in acetylcholine-induced vasodilation in rat isolated perfused kidney. Endothelial denudation by Triton X-100 (0.2%, 0.1 ml) or depletion of afferent nerve endings by capsaicin (10(-6) mol/l) attenuated acetylcholine-induced vasodilation. When these two agents were administered together, the response to acetylcholine was completely inhibited. CGRP1 receptor blocker CGRP 8-37 (10(-7) mol/l) and adenosine A(2) receptor antagonist ZM 241 385 (10(-7) mol/l) inhibited acetylcholine-induced dilation. When indomethacin (10(-5) mol/l), a cyclooxygenase inhibitor, l-NOARG (10(-4) mol/l), a nitric oxide (NO) synthase inhibitor, and potassium chloride (30 mmol/l), to test EDHF response, were perfused simultaneously, the inhibition was greater than that was observed with each agent alone. Guanylate cyclase inhibitor ODQ (10(-5) mol/l) or protein kinase A inhibitor KT 5720 (5x10(-7) mol/l) inhibited acetylcholine-induced dilation. Gap junction uncoupler 18alpha-glycyrrhetinic acid (10(-4) mol/l) caused an uncontrollable increase in basal perfusion pressure making it impossible to test against acetylcholine-induced dilation. Our data suggest that NO, prostanoids, EDHF, and CGRP released from vascular endothelium and afferent nerve endings participate in acetylcholine-induced vasodilation and their signal transduction molecules include protein kinase A and guanylate cyclase.     

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12)to 10(-7)mol/l) was significantly less potent (60-90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10(-7)mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser(2)-Asp(13))GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.     

氨甲酰胆碱和Impromidine对CFU—S细胞周期的影响

正常情况下处于S期的CFU-S比例低于10％。氨甲酰胆碱(Cach 10~(-13)—10~(-9)mol/L)和Impromidine(Impro 10~(-9)—10~(-4)mol/L)在体外与小鼠骨髓细胞短时培育后,增加了CFU-S对细胞毒剂羟基脲的敏感性。反应最大时,9d和13dCFU-S的减少率分别是32.8和60.6％(Cach)以及38.4和49.5％(Impro)。这种效应可分别被胆碱能N受体阻断剂筒箭毒(10~(-6)mol/L)和组胺H_2受体阻断剂甲氰咪呱(10~(-6)mol/L)所阻断,表明9d和13d CFU-S表面胆碱能N受体和组胺H_2受体的密度或活性存在差别,再次证实了CFU-S是一个不均一的细胞群。     

The action of agonists and antagonists at the histamine H1 receptor and receptor protection studies in guinea pig ileum

We have examined the ability of histamine and the competitive reversible antihistamines to protect the histamine H1 receptor against alkylation with the 2-haloalkylamines, phenoxybenzamine and SY-14. In isolated guinea pig ileum these irreversible antagonists produce a parallel shift in the dose-response curve to histamine with retention of the maximum response if they are used at concentrations less than about 10(-6)M. Treatment with these 2-haloalkylamines in the presence of a high concentration of histamine did not alter the blocking activity. Thus histamine appears to be unable to protect its own receptor against irreversible blockade. The competitive reversible antagonists, on the other hand, did provide effective protection against irreversible blockade. It is likely that the competitive reversible H1 receptor antagonists have at least some part of their attachment site in common with irreversible antagonists of the 2-haloalkylamine type, while the inability of histamine to provide self-protection suggests that its primary attachment site is different from that of the antagonists.