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.     

Cholinergic stimulation of isolated rat parietal cells: role of calcium, calmodulin and protein kinase C

We studied the cholinergic stimulation of isolated and enriched rat parietal cells. H+ production was indirectly measured by the uptake of 14C-aminopyrine into the parietal cells. Stimulation by carbachol required the presence of extracellular Ca2+ not only in the initial phase but also during the sustained phase of a 100-min incubation period. The response to carbachol was prevented by the Ca2+ entry blocker lanthanum IC50: 1.5 X 10(-7) mol/l). Furthermore, the dependence on Ca2+ influx of cholinergic stimulation was demonstrated by a 269% increase in total intracellular Ca2+ in response to carbachol, as determined by optical emission spectrometry. The naphthalene sulfonamides W7 and W5 which bind calmodulin and thus block the intracellular transduction of Ca2+ effects also inhibited a carbachol-induced H+ production. In the following experiments we studied the effect of agents which activate the protein kinase C, an enzyme which is supposed to play a key role in intracellular signal transduction of Ca2+-dependent effects. Phospholipase C is supposed to activate protein kinase C via induction of the phosphoinositol breakdown. In our preparation of isolated rat parietal cells, phospholipase C (4-100 mU/ml) exerted inhibition instead of amplification of the response to 10(-4) mol/l carbachol. Similarly, the direct activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate or by 1-oleoyl-2-acetyl-sn-glycerol (both tested at 10(-7) to 10(-5) mol/l) reduced the submaximal and maximal response to 10(-5) or 10(-4) mol/l carbachol. We conclude that the cholinergic stimulation of rat parietal cells is dependent on the influx of extracellular Ca2+. Calmodulin seems to mediate intracellular Ca2+ effects during cholinergic stimulation. The activation of protein kinase C impairs carbachol-induced H+ production instead of augmenting the response. This might be due to an already maximal activation of protein kinase C by carbachol alone or to autoregulatory down-regulation by the protein kinase C of muscarinic parietal-cell receptors.     

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.     

甲硫—脑啡肽对大鼠DRG分离神经元ATP—激活内向电流的抑制

本研究探讨了甲硫-脑啡肽(met-Enk)对ATP-激活电流(IATP)的调制作用.实验在大鼠新鲜分离背根神经节(DRG)神经元上进行.应用全细胞膜片钳技术所记录的IATP为内向电流.在被检测的DRG神经元中,90.0%(45/50)的细胞对ATP有反应.在45个对ATP敏感的细胞中对大部分细胞(29/45)施加met-Enk(10-9～10-5mol/L)也引起一内向电流;少部分细胞(9/45)为外向电流;其余的细胞(7/45)未引起可检测的膜反应.预加met-Enk后IATP明显地被抑制,此种抑制作用为剂量依赖性的.在预加10-9、10-8、10-7、10-6、10-5mol/Lmet-Enk后,IATP的抑制分别为13.2±5.4%(n=5)、39.2±8.6%(n=8)、54.1±8.6%(n=8)、43.3±7.9%(n=7);43.1±7.9%(n=7)(mean±SKM).阿片肽拮抗剂纳洛酮能翻转此种抑制效应.IATP的量-效关系表明,预加met-Enk后曲线明显压低,在浓度为10-3mol/L时IATP下降约25%,而Kd值几乎不变.应用二次钳压技术胞内透析H-9(PKA抑制剂)能取消此种抑制作用.上述结果提示met-Enk对IATP的抑制效应为非竞争性抑制作用,可能是由于阿片受体激活后,经相应的胞内信号转导途径使ATP受体磷酸化所致.     

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.     

Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production.

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurogenic component in the effect of papaverine, drotaverine and bencyclane

Papaverine, drotaverine and bencyclane, drugs considered to have direct action on the smooth muscle, inhibited synaptic transmission in the isolated a sympathetic ganglion of the frog. Their effect depended upon the concentration applied. The ganglionic blocking effect of papaverine and drotaverine in the concentration range from 10(-8) to 10(-6) mol/l was partially antagonized by naloxone and nalorphine as well as by increasing the Ca2+ concentration in the incubation medium. This refers to an activation of specific opiate receptors in the mechanism of ganglionic action of these drugs. The ganglionic effect of bencyclane may be due to its local anaesthetic property, since it was prevented by neither naloxone nor nalorphine, and an increase in the Ca2+ concentration in the medium had no influence on it.     

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide,amiloride and omeprazole

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.     

γ-氨基丁酸对小白鼠离体胃标本胃酸分泌的促进效应

为了探索γ-氨基丁酸(GABA)对小白鼠离体胃标本胃酸分泌(GAS)的影响及机制,在体外37℃缓冲液中培育离体、胃腔灌流并维持胃内12 cm水柱压力的全胃标本,用pHS-3型精密酸度计测定灌流液的pH。结果表明：γ-氨基丁酸(GABA)(1～10×10-7mol/L)和巴氯芬(Bac, 0.6～9.6×10-7mol/L)以一种浓度依赖的方式显著地促进胃酸分泌(GAS),而西咪替丁(Cim, 2～20×10-7mol/L)以一种浓度依赖的方式有力地抑制GAS。印防已毒素(Pic, 3×10-7mol/L)不影响基础胃酸分泌(BGAS)和GABA促进GAS的效应,而番氯芬(Phac, 0.6×10-7mol/L)能完全阻断GABA的促进效应。Cim不能完全消除GABA和Bac对GAS的促进效应。以上结果提示,在小鼠中GABA可以通过激活胃中GABAB受体促进离体胃标本的GAS,可能胃壁胆碱能神经元和非神经细胞,如壁细胞及某些内分泌细胞上都存在GABAB受体,GABA可直接或简接地剌激胃壁细胞分泌酸。     

Control of sulfatase and sulfotransferase activities by medrogestone in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines.

In the present study, we explored the effect of the progestin medrogestone on the sulfatase and sulfotransferase activities in the hormone-dependent MCF-7 and T-47D human breast cancer cell lines. After 24 h incubation at 37 degrees C of physiological concentrations of estrone sulfate ([3H]-E1S: 5x10(-9) mol/l), it was observed that this estrogen was converted in a great proportion to E2 in both cell lines. Medrogestone significantly inhibits this transformation, at all the concentrations tested (5x10(-8) to 5x10(-5) mol/l), in both cell lines. The IC50 values were 1.93 micromol/l and 0.21 micromol/l in MCF-7 and T-47D cells, respectively. In another series of studies, after 24 h incubation at 37 degrees C of physiological concentrations of estrone ([3H]-E1: 5x10(-9) mol/l), the sulfotransferase activity was detectable in both cell lines. Estrogen sulfates (ES) are found exclusively in the culture medium, which suggests that as soon as they are formed they are excreted into the medium. Medrogestone has a biphasic effect on sulfotransferase activity in both cell lines. At low doses: 5x10(-8) and 5x10(-7) mol/l, this compound stimulates the enzyme by +73.5 and 52.7%, respectively, in MCF-7, and by 84.5 and 62.6% in T-47D cells. At high concentrations: 5x10(-6) and 5x10(-5) mol/l, medrogestone has no effect on MCF-7 cells, but inhibits the sulfotransferase activity in T-47D cells by -31.4% at 5x10(-5) mol/l. In conclusion, the inhibitory effect provoked by medrogestone on the enzyme involved in the biosynthesis of E2 (sulfatase pathway) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive ES, support a probable anti-proliferative effect of this progestin in breast tissue. Clinical applications of these findings can open new therapeutic possibilities for this disease.     

Failure of opiates to increase the hydrolysis of GTP in neuroblastoma-glioma 108-15 cells

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low K_m GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [³H]guanine followed by incubation with 10^?6M guanine. Etorphine (10^?6M] or vehicle were added and the incubations continued for 1–4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE₁. We also studied the effects of naloxone and PGE₁ on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.     

Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.     

Atrial natriuretic peptide relaxes arterial basal tone induced by coarctation hypertension.

We have investigated the vasorelaxant effect of atrial natriuretic peptide (ANP) on isolated non-contracted aorta from coarctation hypertensive rats (HR) and the role of endothelium in this vasorelaxant action. After 7-14 days of surgery, mean blood pressure was higher (P < 0.01) in HR compared with sham operated rats (SR), used as the control. ANP (10(-6) mol/l) significantly lowered basal tone in previously unstimulated HR thoracic aortic rings; however, it had no effect in HR abdominal aorta or in SR abdominal and thoracic aorta. Endothelial destruction potentiated the vasorelaxant effect of ANP on basal tone in HR thoracic aorta. A similar potentiation of the ANP-response was observed by pre-treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) mol/l) or methylene blue (2 x 10(-5) mol/l) in unrubbed HR thoracic aorta. Treatment with calcium-free Krebs + EGTA (2 x 10(-3) mol/l) + sodium nitroprusside (10(-5) mol/l) or calcium-free Krebs significantly decreased basal tone and abolished ANP-response. These effects were observed only in HR thoracic aorta. Similarly, staurosporine (10(-7) mol/l) and calphostin C (10(-6) mol/l), inhibitors of protein kinase C (PKC), diminished basal tone and abolished the ANP-response in HR thoracic aorta. Acetylcholine (10(-6) mol/l) had a small but significant action on the basal tone of unrubbed HR thoracic aorta. These results demonstrate that ANP has a vasorelaxant effect on aortic basal tone when the vessel is exposed to high blood pressure. Inhibition of ANP effects on basal tone by calcium-free Krebs and PKC antagonists suggests that the HR aorta increases Ca2+-active tone, that modifies the response of vascular smooth muscle to the vasodilating hormone ANP.     

Opioid involvement in the control of feeding in an insect, the American cockroach

Administration of the kappa opiate agonist, U-50,488H (0.10-10 mg/kg), produced over three hours a significant dose-dependent increase in the ingestive responses of free feeding American cockroaches, Periplaneta americana. These effects could be decreased by the opiate antagonist, naloxone (1.0 mg/kg), with naloxone by itself blocking the augmented feeding responses of food-deprived cockroaches. The mu opiate agonist, morphine (1.0-20 mg/kg) caused a significant dose-dependent and naloxone-reversible increase in the locomotory activity of cockroaches. These results suggest that opioid systems may be involved in the control of the feeding in cockroaches in a manner analogous to that proposed for vertebrates.     

Kappa- but not delta-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats

Two series of experiments were performed in the isolated perfused rat heart to determine the role of kappa- and delta-opioid receptors (OR) in cardioprotection of ischemic preconditioning (IP). In the first series of experiments, it was found that IP with two cycles of 5-min regional ischemia followed by 5-min reperfusion each reduced infarct size induced by 30-min ischemia, and the ameliorating effect of IP on infarct was attenuated with blockade of either 5 x 10(-6) mol/l nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, or 5 x 10(-6) mol/l naltrindole (NTD), a selective delta-OR antagonist. The second series showed that U50,488H, a selective kappa-OR agonist, or D-Ala(2)-D-leu(5)-enkephalin (DADLE), a selective delta-OR agonist, dose dependently reduced the infarct size induced by ischemia, which mimicked the effects of IP. The effect of 10(-5) mol/l U50,488H on infarct was significantly attenuated by blockade of protein kinase C (PKC) with specific PKC inhibitors, 5 x 10(-6) mol/l chelerythrine or 8 x 10(-7) mol/l calphostin C, as well as by blockade of ATP-sensitive K(+) (K(ATP)) channels with blockers of the channel, 10(-5) mol/l glibenclamide or 10(-4) mol/l 5-hydroxydecanoate. IP also reduced arrhythmia induced by ischemia. Nor-BNI, but not NTD, attenuated, while U50,488H, but not DADLE, mimicked the antiarrhythmic action of IP. In conclusion, the present study has provided first evidence that kappa-OR mediates the ameliorating effects of IP on infarct and arrhythmia induced by ischemia, whereas delta-OR mediates the effects only on infarct. Both PKC and K(ATP) channels mediate the effect of activation of kappa-OR on infarct.     

Production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1, by cultured human adrenocortical carcinoma cells

The production and secretion of peptides by adrenocortical tumors have not been well studied. We therefore studied the production and secretion of two vasoactive peptides, adrenomedullin and endothelin-1 in SW-13 human adrenocortical carcinoma cells by radioimmunoassay and Northern blot analysis. Both immunoreactive-adrenomedullin and immunoreactive-endothelin were detected in the culture medium of SW-13 cells (27.7 +/- 1.6 fmol/10 (5) cells/24 h and 11.0 +/- 0.8 fmol/10 (5) cells/24 h, respectively, mean +/- SEM, n = 6). Northern blot analysis showed the expression of adrenomedullin mRNA and endothelin-1 mRNA in SW-13 cells. On the other hand, no significant amount of calcitonin gene-related peptide, corticotropin-releasing hormone, neuropeptide Y, or urocortin was secreted by SW-13 cells. Treatment with ACTH (10(-9)-10(-7) mol/l), angiotensin II (10(-9)-10(-7) mol/l), or dexamethasone (10(-8)-10(-6) mol/l) for 24 h had no significant effects on immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium of SW-13. Treatment with tumor necrosis factor (TNF)-alpha (20 ng/ml) increased significantly both immunoreactive-adrenomedullin levels and immunoreactive-endothelin levels in the culture medium. Interferon-gamma (100 U/ml) increased the immunoreactive-endothelin levels, but not immunoreactive-adrenomedullin levels, whereas interleukin-1 (IL-1)beta (10 ng/ml) increased immunoreactive-adrenomedullin levels, but not immunoreactive-endothelin levels. These findings indicate that SW-13 human adrenocortical carcinoma cells produce and secrete two vasoactive peptides, adrenomedullin, and endothelin-1 and that the secretion of these two peptides is modulated differently by cytokines.     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

Nalorphine: actions at an opiate receptor on frog skeletal muscle fibers

Intracellular microelectrode studies were conducted to investigate the actions of the partial agonist-antagonist nalorphine at an opiate receptor on functional frog skeletal muscle fiber membranes. In high bath concentrations (greater than or equal to 10(-4) M), nalorphine alone produces agonist actions similar to the "full" opiate agonists. These actions were (i) to depress both the sodium and potassium (gNa and gK) conductance increases due to electrical stimulation by a nonspecific local anestheticlike mechanism and (ii) to depress gNa by a specific opiate receptor mediated mechanism. In a much lower bath concentration (1 X 10(-8) M) nalorphine acts to antagonize the specific opiate receptor mediated depression of gNa produced by the "full" agonist meperidine. Thus in this preparation nalorphine, "the partial antagonist," has the same actions as naloxone, which is often considered to be a full antagonist. The quantitative differences observed in the effects of these two opiate antagonists are discussed.     

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Barley limit dextrinase (HvLD) of glycoside hydrolase family 13 is the sole enzyme hydrolysing α-1,6-glucosidic linkages from starch in the germinating seed. Surprisingly, HvLD shows 150- and 7-fold higher activity towards pullulan and β-limit dextrin, respectively, than amylopectin. This is investigated by mutational analysis of residues in the N-terminal CBM-21-like domain (Ser14Arg, His108Arg, Ser14Arg/His108Arg) and at the outer subsites +2 (Phe553Gly) and +3 (Phe620Ala, Asp621Ala, Phe620Ala/Asp621Ala) of the active site. The Ser14 and His108 mutants mimic natural LD variants from sorghum and rice with elevated enzymatic activity. Although situated about 40 Å from the active site, the single mutants had 15–40% catalytic efficiency compared to wild type for the three polysaccharides and the double mutant retained 27% activity for β-limit dextrin and 64% for pullulan and amylopectin. These three mutants hydrolysed 4,6-O-benzylidene-4-nitrophenyl-6³-α-d-maltotriosyl-maltotriose (BPNPG3G3) with 51–109% of wild-type activity. The results highlight that the N-terminal CBM21-like domain plays a role in activity. Phe553 and the highly conserved Trp512 sandwich a substrate main chain glucosyl residue at subsite +2 of the active site, while substrate contacts of Phe620 and Asp621 at subsite +3 are less prominent. Phe553Gly showed 47% and 25% activity on pullulan and BPNPG3G3, respectively having a main role at subsite +2. By contrast at subsite +3, Asp621Ala increased activity on pullulan by 2.4-fold, while Phe620Ala/Asp621Ala retained only 7% activity on pullulan albeit showed 25% activity towards BPNPG3G3. This outcome supports that the outer substrate binding area harbours preference determinants for the branched substrates amylopectin and β-limit dextrin.