Actions of gastrin-releasing peptide and related mammalian and amphibian peptides on ion transport in the porcine proximal jejunum

The 27-amino acid peptide gastrin-releasing peptide (GRP) and the decapeptide neuromedin B (NMB) are structurally related to bombesin (BB) and exist within the mammalian small intestine. We examined the actions of porcine GRP and NMB on ion transport in the porcine proximal jejunum in vitro and compared their activities to those of their respective C-terminal amphibian homologs BB and ranatensin (RT). The 4 peptides transiently increased potential difference and short-circuit current (Isc) in jejunal mucosal sheets after their serosal administration in subnanomolar concentrations with an order of potency: GRP approximately RT greater than or equal to NMB greater than BB. BB and RT were more effective in elevating Isc than GRP and NMB; all peptides had variable effects on tissue conductance. Mucosal Isc responses to GRP (1 nM) were due in part to a stimulation of net Cl- secretion. GRP-induced Isc increases were halved by serosal furosemide (0.3 mM) and reduced by 65% and 90% in tissue bathing solutions lacking Cl- or Cl- and HCO3-, respectively. Tetrodotoxin reduced Isc responses to the peptide by 40%; GRP activity remained unaffected after blockade of gut muscarinic or nicotinic cholinergic receptors by atropine or hexamethonium, respectively. These results suggest that GRP and its natural homologs stimulate active electrogenic Cl- secretion in the porcine jejunum through interactions with GRP receptors located in the intestinal mucosa and submucosa.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

神经肽Y对心室肌细胞离子通道的影响

采用全细胞膜片钳技术观察神经肽Y(neuropeptide Y,NPY)对心室肌细胞离子通道的影响。结果如下：(1)NPY浓度在1．0～100nmol/L范围内剂量依赖性抑制大鼠心室肌细胞I_(Ca-L),IC_(50)值为1．86nmol/L。NPY对I_(Ca-L)的I-V曲线的最大峰值电位、激活和失活电位均无显著影响。NPY对去甲肾上腺素(norepinephrine,NE)增加的I_(Ca-L)有显著抑制作用。(2)NPY对人鼠心室肌细胞I_(Na/Ca)有显著抑制作用。10nmol/L NPY使前向I__(Na/Ca)由(0．27±0．11)pA/pF减小为(0．06±0．01)pA/pF;反向I__(Na/Ca)由(0．45±0．12)pA/pF降为(0．27±0．09)pA/pF(P＜0．05,n=4)。(3)NPY对大鼠心室肌细胞I_(to)有显著增强作用。10 nmol/L NPY使I_(to)由(12．5±0．70)pA/pF增加至(14．7±0．59)pA/pF(P＜0．05,n=4)。(4)10nmol/L NPY对大鼠心室肌细胞I_(Na)没有显著影响。(5)10nmol/L NPY对豚鼠心室肌细胞I_K无明显影响。研究结果证实,NPY抑制大鼠心室肌细胞I_(Ca-L)和I_(Na/Ca),增强I_(to)对I_Na和豚鼠心审肌细胞I_K没有显著作用,表明NPY对上述主要离子通道的效应与NE的效应相拮抗。     

Synthesis of porcine neuropeptide Y(NPY) in solution

The porcine neuropeptide Y (NPY), a 36-residue peptide amide, was synthesized by assembling six peptide fragments followed by thioanisole-mediated deprotection with trifluoromethanesulfonic acid in trifluoroacetic acid. beta-Cycloheptyl aspartate, Asp(OChp), was employed to suppress base-catalyzed succinimide formation. When administered to dogs, the purified peptide (10 micrograms/kg) caused prolonged increase of systemic arterial blood pressure and decreased pancreatic blood flow.     

Modeling of neuropeptide receptors Y1, Y4, Y5, and docking studies with neuropeptide antagonist

Neuropeptide Y (NPY), receptors belong to the G-protein coupled receptor superfamily. NPY mediates several physiological responses, such as blood pressure, food intake, sedation. These actions of NPY are mediated by six receptor subtypes denoted as Y1-Y5 and y6. Modeling of receptor subtypes and binding site identification is an important step in developing new therapeutic agents. We have attempted to model the three NPY receptor types, Y1, Y4, and Y5 using homology modeling and threading methods. The models are consistent with previously reported experimental evidence. To understand the interaction and selectivity of NPY analogues with different neuropeptide receptors, docking studies of two neuropeptide analogues (BVD10 and BVD15) with receptors Y1 and Y4 were carried out. Results of the docking studies indicated that the interaction of ligands BVD10 and BVD15 with Y1 and Y4 receptors are different. These results were evaluated for selectivity of peptide analogues BVD10 and BVD15 towards the receptors.     

Design of the Y1-receptor-selective cyclic peptide based on the C-terminal sequence of neuropeptide Y.

We designed four cyclic peptides which are mimics of the C-terminal region of human neuropeptide Y (NPY) on the basis of the structural model of NPY. One of these cyclic peptides, c[D-Cys29-L-Cys34]NPY Ac-29-36 (YM-42454), exhibited significantly higher affinity for the Y1-receptor than the corresponding C-terminal linear fragment, NPY Ac-28-36. Interestingly, YM-42454 showed binding affinity for the Y1-receptor in spite of the lack of the N-terminal sequence of NPY, whereas it did not show any binding affinity for the Y2-receptor. This conformationally restricted Y1-selective peptide would provide some insights into the bioactive conformation of the C-terminal region of NPY.     

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.     

The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa

The antisecretory potency of NPY and a series of truncated and structural analogues of NPY have been tested upon mucosal preparations of rat small intestine. Single amino acid substitutions, i.e., [Ile34]NPY, [Pro34]NPY, resulted in severe attenuation and loss of biological activity, respectively, and neither peptide affected NPY responses. An agonist order of potency: NPY greater than or equal to [Glu16,Ser18,Ala22,Leu28,31]NPY (ESALL-NPY) greater than [Cys2,Aoc5-24,DCys27]NPY (C2-NPY) greater than [Aoc5-24]NPY greater than [Des-Ser3,Des- Lys4]C2-NPY much greater than [Cys5,Aoc7-20,DCys24]NPY (C5-NPY) greater than equal to [DCys7,Aoc8-17, Cys20]NPY (C7-NPY) greater than [Aoc8-17]NPY greater than or equal to [Ile34]C7-NPY much greater than [Aoc2-27]NPY much greater than [Pro34]C2-NPY was obtained. The use of analogues based upon the tertiary structural model of NPY with varying amounts of N- and C-terminal helical regions removed and replaced with a single 8-aminooctanoic acid residue (Aoc) has allowed us to assess the structural requirements for activation of the regions in close apposition to each other. The polyproline helix, beta-turn and majority of the amphipathic alpha-helix serve a structural role bringing N- and C-terminal residues together for optimal receptor recognition and activation.     

Insatiable feeding evoked in rats by recurrent perfusion of neuropeptide Y in the hypothalamus

Hyperphagic-like intake of food was determined in the unrestrained rat during the sustained elevation over time of neuropeptide Y (NPY) within the paraventricular nucleus (PVN) and surrounding hypothalamic regions. A single guide tube was implanted stereotaxically in each of 22 rats for localized, intermittent perfusions of a CSF vehicle, nondeprotected NPY(1-36) or native NPY. Each site in the PVN of the fully sated rat was perfused repeatedly over a 5.0-h interval by means of a standard push-pull cannula system at a rate of 20 microliters/min for 6.0 min in one of three concentrations: 0.2, 1.0 and 2.0 micrograms/min. Two perfusions of 1.0 micrograms/min NPY evoked an intake of 4.6 +/- 1.1 g of food over a 3.0-h period, whereas 4-7 and 8-15 perfusions of this concentration of NPY, distributed over 5.0 h, induced the sated rats to eat a total of 12.0 +/- 1.1 g and 33.2 +/- 3.0 g, respectively. During a fixed number of 10 hypothalamic perfusions distributed over 5.0 h, concentrations of 0.2 and 2.0 micrograms/min NPY caused a cumulative intake of food in the rats of 14.2 +/- 2.0 g and 31.7 +/- 3.3 g, respectively. Under each condition, parallel push-pull perfusions of either control solution in the same hypothalamic sites were without effect on feeding. During the 5.0-h interval of repeated perfusions, successive bouts of eating occurred with individual intakes of food reaching as high as 49.0 g, which exceeded by up to two-fold the entire daily consumption of food. However, ingestion of water was unaffected by perfusion of NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

Storage and fast transport of noradrenaline, dopamine beta-hydroxylase and neuropeptide Y in dog sciatic nerve axons.

The axonal transport and subcellular distribution of noradrenaline (NA), dopamine beta-hydroxylase (DBH) and neuropeptide Y (NPY) were determined in dog sciatic nerve using an accumulation technique. The results were compared with those obtained by application of the same procedures and methods on the splenic nerve in the same animal species. Evidence was found for the coexistence of NA and NPY in large dense-cored vesicles in dog sciatic nerve axons. After differential centrifugation and isopyenic sucrose density gradient centrifugation of 24 h ligated sciatic nerve pieces NA and NPY equilibrated around 1M sucrose. The DBH activity was dispersed broadly on the gradient. Subsequently, the accumulation of NA, DBH and NPY was studied in proximal and sital segments of 8, 12 and 24 h dog ligated sciatic nerve and inferences were made concerning the axonal transport of these compounds. NA, DBH and NPY displayed a divergent accumulation proximal to the ligation. After 12 h of ligation a transport rate was calculated of 4.8 +/- 1.8 mm/h for NA, of 5.9 +/- 1.5 mm/h for DBH and of 4.9 +/- 2.0 mm/h for NPY. With a correction for the stationary fractions, a similar fast transport rate of approximately 10 to 12 mm/h was proposed for NA, DBH and NPY. The occurrence was shown of a limited retrograde transport of DBH and possibly NPY, but not of NA.     

Role of adrenal hormones in regulating distal nephron structure and ion transport

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.     

Localization of neuropeptide Y and norepinephrine in the porcine ovarian artery and their influence on the local blood pressure

The aim of the present study was to determine: (i) the presence of dopamine-beta-hydroxylase (DbetaH)- and neuropeptide Y (NPY)-immunoreactive (IR) nerve fibres in the wall of the porcine ovarian artery, (ii) the influence of NPY and norepinephrine (NE) on the contractile activity of the pig ovarian arteries, and (iii) the pharmacological analysis of the interaction between NPY and NE in the isolated porcine ovarian arteries collected from immature pigs and from animals in different days of the estrous cycle. Ovarian arteries for immunohistochemistry and isolated arteries for pharmacological studies were excised from immature pigs and mature animals on days 1-5, 8-13 and 17-20 of the estrous cycle. The study showed that both DbetaH- and NPY-IR nerve fibres were present in the pig ovarian arteries in all periods examined, and, that in some fibres DbetaH and NPY were co-localized. Both NE (10(-6) M) and NPY (10(-7) M) increased blood pressure of examined preparations, however, NE caused stronger changes in the vessel wall tension (P<0.001), than did NPY. NE significantly increased (P<0.001) blood pressure of all isolated arteries, however, this response was stronger in vessels from days 1-5 of the cycle, when compared to days 8-13 (P<0.01), 17-20 and immature pigs (P<0.001). NPY increased significantly blood pressure in isolated arteries from days 8-13 and 17-20 of the cycle (P<0.001), while in preparations taken from immature pigs and animals on days 1-5 of the estrous cycle this response was somewhat weaker (P<0.01). A higher elevation (P<0.001) of blood pressure after NPY administration was observed in isolated arteries from days 17-20 of the cycle, when compared to vessels from days 1-5 and 8-13 and those from immature pigs. Moreover, NE significantly intensified (P<0.001) an increase in the blood pressure in isolated arteries pre-treated with NPY in all periods examined. NPY insignificantly (P>0.05) potentiated increase of blood pressure in NE pre-treated vessels of immature pigs and in isolated arteries from days 17-20 of the cycle and significantly (P<0.05) in vessels from days 1-5 and 8-13 of the estrous cycle. Our results indicate that DbetaH- and NPY-IR nerve fibres are present in the pig ovarian arteries. NE and NPY administered alone increased blood pressure in the pig isolated ovarian artery and simultaneous administration of both substances caused each other potentiation of vasocontractile effect, however, the strength of observed changes was dependent on the stage of the estrous cycle.     

Design, synthesis, conformational and membrane ion transport studies of proline-adamantane hybrid cyclic depsipeptides

The design, synthesis, conformational, crystallographic, and ion transport studies of 30-membered, proline containing depsipeptides that incorporate the rigid low molecular weight lipophilic adamantane (Adm) building blocks are reported. The adamantyl groups provide the desired membrane permeability and conformational constraint for efficient transport in lipid membranes. The novel cyclic depsipeptides are: c[--Adm--C(O)--Pro-- O--CH(2)-- CHR--NH--C(O)--Pro--C(O)-- Adm--C(O)--Pro--C(O)--NH--CHR--CH(2)-- O--Pro--C(O)--] where R==H for A and R==CONH--Adm for B. Crystal structure analysis of A established that the two peptide segments are identical in formula and in conformation and that the peptides are bonded to the interleaving Adm at the 1 and 3 positions. However, the complete ring is highly asymmetric in shape since bonds for both Peptide-Adm-Peptide segments have the syn-anti motif. Torsional angles for the connecting bonds to Adm are -162 degrees , +71 degrees and -169 degrees , -48 degrees . The irregular clamshell shape of the molecule has three internal C==O moieties directed in a manner that could provide three Na(+)--O ligands. While A exhibited negligible transport of Na(+) ions across membranes, peptide B endowed with two additional adamantanes in the periphery did transport Na(+) ions from outside to inside.     

Mode of action of ANG II on ion transport in guinea pig distal colon

The effect of ANG II on mucosal ion transport and localization of ANG type 1 receptor (AT(1)R) in the guinea pig distal colon was investigated. Submucosal/mucosal segments were mounted in Ussing flux chambers, and short-circuit current (I(sc)) was measured as an index of ion transport. Serosal addition of ANG II produced a concentration-dependent (10(-9)-10(-5) M) increase in I(sc). The maximal response was observed at 10(-6) M; the increase in I(sc) was 164.4 +/- 11.8 microA/cm(2). The ANG II (10(-6) M)-evoked response was mainly due to Cl(-) secretion. Tetrodotoxin, atropine, the neurokinin type 1 receptor antagonist FK-888, and piroxicam significantly reduced the ANG II (10(-6) M)-evoked response to 28, 45, 58, and 16% of control, respectively. Pretreatment with prostaglandin E(2) (10(-5) M) resulted in a threefold increase in the ANG II-evoked response. The AT(1)R antagonist FR-130739 completely blocked ANG II (10(-6) M)-evoked responses, whereas the ANG type 2 receptor antagonist PD-123319 had no effect. Localization of AT(1)R was determined by immunohistochemistry. In the immunohistochemical study, AT(1)R-immunopositive cells were distributed clearly in enteric nerves and moderately in surface epithelial cells. These results suggest that ANG II-evoked electrogenic Cl(-) secretion may involve submucosal cholinergic and tachykinergic neurons and prostanoid synthesis pathways through AT(1)R on the submucosal plexus and surface epithelial cells in guinea pig distal colon.