P物质放射免疫测定法及在大白鼠动情周期中垂体内P物质含量的变化

本文报道了P物质放射免疫测定方法的建立。用此方法测定了大白鼠脑不同部位(下丘脑、垂体、嗅球和小脑)中P物质的含量,还测定了大白鼠不同情期垂体中P物质含量的变化,观察到间情期垂体P物质的含量最高,动情前期显著降低,动情期仍维持在低水平。因此认为P物质在垂体水平对LH和FSH的分泌有抑制性影响。     

The effects of substance P and met5-enkephalin in dog ileum

Substance P initiated tonic contraction of dog ileum when administered in doses from 1 pg to 20 micrograms intraarterially (ED50 = 67 ng). Low doses acted to excite cholinergic postganglionic neurones since atropine or tetrodotoxin (TTX) increased the ED50 of substance P about 25-fold, while hexamethonium and local field stimulation had only a small effect to increase the ED50. Also atropine and tetrodotoxin effects were not additive. Higher doses apparently acted to stimulate smooth muscle directly, but no evidence was obtained that local field stimulation could release substance P to act on smooth muscle. Substance P tachyphylaxis prevented substance P actions on cholinergic nerves, but it did not affect responses to intraaterial acetylcholine or block distal inhibition from proximal distention or field stimulation. Met-enkephalin given intraarterially, was also excitatory in doses from 1 ng to 20 micrograms; the amplitude of tonic and phasic contractions produced was significantly decreased by TTX and atropine but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced but was not diminished by hexamethonium or substance P tachyphylaxis. Partial tachyphylaxis to met-enkephalin was produced without affecting the ED50 for substance P. We conclude that substance P acts in small amounts on receptors in myenteric nerves to release acetylcholine by a mechanism, presumably involving postganglionic cholinergic nerves, while met-enkephalin also apparently may act at least in part through a similar TTX- and atropine-sensitive mechanism. These peptides also caused activation of other receptors, probably on smooth muscle by noncholinergic. TTX-insensitive mechanisms. Also the receptors for each peptide which are located on nerves were distinct and independent since tachyphylaxis could be produced to each without affecting the response to the other.     

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.     

Renal metabolism of substance P in the dog.

Substance P is a potent vasodilatory, diuretic, and natriuretic agent. Since subcellular fractions of the kidney rapidly inactivate substance P in vitro, the present study was designed to examine this observation $\text{in}$$\text{vivo}$ in anesthetized dogs. Arterial, renal venous, and urinary levels of immunoreactive substance P were determined by radioimmunoassay and were found to be 117±11, 128±12 and 659±104 pg/ml, respectively. The urinary and fractional excretion of immunoreactive substance P were 122±22 pg/min and 6.6±2.0%, respectively. When substance P was infused intravenously, the arterial and renal venous plasma levels of immunoreactive material increased whereas the urinary levels did not change. Infusions of 50 ng/kg/min of substance P significantly decreased mean arterial pressure, urinary volume, creatinine clearance as well as the urinary excretion, clearance, and fractional excretion of immunoreactive substance P. During intrarenal infusion of ¹²⁵I-(8-Tyr) substance P, high levels of radioactive material were found in the urine and renal venous plasma which failed to migrate on thin layer chromatography with intact ¹²⁵I-(8-Tyr) substance P. Thus under these conditions, intact substance P was not released from the kidney into the urine or renal venous blood, but instead circulating substance P was rapidly and completely metabolized, probably by both vascular and tubular elements of the kidney.     

Substance P and neurokinin A are codistributed and colocalized in the porcine gastrointestinal tract.

Immunoreactive substance P and neurokinin A were measured with radioimmunoassay in extracts of different segments of porcine gastrointestinal tract using C-terminally directed antisera. In all segments, the concentrations of substance P and neurokinin A were similar. The largest concentrations of both peptides were found in the mid-colon. By gel chromatography and reversed-phase high pressure liquid chromatography the immunoreactivity in extracts from ileum eluted as homogenous peptides at the positions of synthetic substance P and neurokinin A, respectively. No neurokinin B was found. By immunohistochemistry of porcine duodenum, jejunum, ileum and mid-colon, identical localization patterns were found for substance P and neurokinin A, and the two peptides demonstrated by double immunofluorescence to be colocalized in the enteric nervous system of the ileum. We conclude that the tachykinins substance P and neurokinin A are codistributed and colocalized in the procine gastrointestinal tract and suggest that the two peptides are produced from a common precursor, beta- and/or gamma-preprotachykinin, in the same neurons.     

Avidin binding of biotinylated analogues of substance P

We report the bioactivities of three biotinylated analogues of Substance P, [alpha-biotinyl-Lys3]Substance P-(3-11), [alpha-biotinyl-Arg1]Substance P, [epsilon-biotinyl-Lys3]Substance P, as well as the bioactivities of their complexes with avidin on guinea pig ileum. The rate of dissociation of an [alpha-biotinyl-Arg1]Substance P-avidin complex has been determined in the presence of 2-(4'-hydroxyazobenzene) benzoic acid and [3H]biotin. The biphasic dissociation of a 4:4 complex between [alpha-biotinyl-Arg1]Substance P and avidin led us to postulate the existence of two sets of binding sites, with the following rates of dissociation, at 4 degrees C, kappa-1 = 6 x 10(-4) x s-1 and kappa-1 = 1.2 x 10(-5) x s-1. We have confirmed this non-equivalence of the four binding sites of avidin by nuclear magnetic resonance using a spin-echo technique. The [alpha-biotinyl-Arg1]Substance P-avidin may be used for the affinity chromatography of Substance P receptors and the decreased affinity of this complex may be taken as an advantage since it can be displaced under mild conditions, i.e. by biotin-containing buffer.     

Conversion of substance P to C-terminal fragments in human plasma

Substance P is rapidly converted by enzyme(s) in human plasma to des-[Arg1Pro2]-substance P (fragment 3-11) and to des-[Arg1Pro2Lys3Pro4]-substance P (fragment 5-11). These metabolites were isolated by HPLC and partially sequenced. No evidence was obtained for deamidation of substance P in plasma or for the formation of the N-terminal tetrapeptide [Arg-Pro-Lys-Pro]. The data suggest that substance P is metabolized in human plasma by an enzyme with the specificity of dipeptidyl-aminopeptidase IV. Consistent with this hypothesis, the rate of degradation of substance P measured with an antibody directed against the N-terminal region is 2-3-fold greater than measured with a C-terminally directed antibody. The degrading activity of plasma was purified 522-fold and was eluted from a gel filtration column in the molecular weight zone 150 000-170 000 and from a chromatofocusing column in the pH range 4.5 to 5.5.     

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.     

Active Uptake of Substance P Carboxy-Terminal Heptapeptide (5–11) into Rat Brain and Rabbit Spinal Cord Slices

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.     

Participation of substance P in the control of renin release.

The effect of the hypothalamic undecapeptide substance P on renin secretion rate was studied in the denervated dog kidney. Intrarenal infusion of substance P at a rate of 0.2 ng/kg/min suppressed renin secretion rates from 258.5 ± 28.5 ng/min to 133.1 ± 23.2 ng/min (p<0.001). Substance P infused at this dose neither changed blood pressure nor did it affect renal cortical plasma flow, glomerular filtration rate or sodium excretion. Thus, the suppression of renin release by substance P cannot be explained by any of the known control mechanisms. It is proposed that substance P participates in the control of renin release by a direct effect on the juxtaglomerular cells.     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Autoradiographic analysis of binding sites for 125I-Bolton-Hunter-substance P in the human eye.

Substance P is known to exert potent effects in peripheral tissues, and is thought to be important for ocular function. The mechanism of action of substance P in the human eye is not known. As a basis for biochemical characterization specific binding of 125I-Bolton-Hunter-substance P was demonstrated in the human eye using autoradiographic methods. Biochemical characterization on slide-mounted tissue preparations showed that binding was saturable with a KD of 0.27 +/- 0.1 nmol/l. Specific binding occurred at comparable autoradiographic densities to both human retina and choroid. Substance P and its carboxyterminal fragment, substance P(3-11), were shown to be highly potent in binding competition experiments against 125I-Bolton-Hunter-substance P. Similar concentrations of substance P(1-9), neurokinin A and neurokinin B failed to significantly alter specific binding of 125I-Bolton-Hunter-substance P. The results indicate expression of high affinity substance P binding sites in human retina and choroid.     

Effects of Substance P on Nicotinic Acetylcholine Receptor Function in PC 12 Cells

The effects of substance P on the functioning of nicotinic acetylcholine receptors in PC12 cells were examined. Carbachol-stimulated 22Na+ uptake was used to assess the functional state of the nicotinic receptor. We found that incubation of the cells with substance P alone caused a loss of receptor function. Receptors recovered from this effect with a t1/2 of 0.94 +/- 0.10 min. Since receptors recovered from carbachol-induced desensitization at a significantly slower rate (t1/2, 1.77 +/- 0.21 min), it was concluded that the two inactive states are not kinetically equivalent. The effects of substance P on carbachol-induced loss of receptor activity were also examined. Substance P had no effect on a component of carbachol-induced loss of activity that was nonrecoverable (inactivation). However, substance P had several effects on the recoverable loss of activity induced by carbachol (desensitization). Substance P caused a shift to the left in the EC50 for carbachol-induced desensitization at equilibrium. If cells were simultaneously incubated with carbachol and substance P7-11, a low-potency analog of substance P, an increase in the rate of formation of a state of the receptor that was kinetically indistinguishable from the state induced by carbachol alone was observed. However, not all inhibition of nicotinic cholinergic function could be explained by an increased rate of formation of a desensitized receptor and it is concluded that substance P causes both enhanced desensitization and block of the nicotinic receptor-linked channel.     

Distinct classes of substance P receptors revealed by a comparison of the activities of substance P and some of its segments

The contracting potency of Substance P and of its C-terminal fragments was studied using four isolated preparations of smooth muscle. The Substance P receptors in the four muscles studied can be differentiated on the basis of their interactions with Substance P and its C-terminal fragments. On the guinea pig ileum, the potency of Substance P is equal to that of the C-terminal octa- and heptapeptide segments and in the rat ileum the potency of Substance P is equal to that of the C-terminal octapeptide and even higher than that of the heptapeptide. In contrast, on the cow pupillary sphincter and guinea pig urinary bladder, Substance P is markedly less potent that the C-terminal octa-, hepta- and hexapeptides. These results suggest the existence of different classes of Substance P receptors and indicate that the N-terminal sequence may be important in regulating Substance P activity.     

P物质的免疫调节作用

P物质在外周主要分布于了发出细传入的神经元内。在外周神经末梢释放的P物质参与免疫调节和炎症过程。P物质可以影响淋巴细胞的增殖、免疫球蛋白和细胞因子的合成,并能够调节辅佐细胞的活性和细胞因子的合成以及其他一些免疫细胞的活性。P物质通过以上作用参与调节细胞和体液免疫应答。在外周组织中,P物质能的神经纤维和一些免疫细胞联系密切,许多免疫细胞膜上存在有P物质的特异性受体。这些形态学资料为P物质参与免疫调节提供了证据。一些免疫细胞也能够产生P物质,并以自分泌或／和旁分泌的方式调节免疫细胞的功能。以上资料表明P物质不仅是一种神经肽,也是一种免疫调节因子,是神经系统和免疫系统共同的信使物质。     

Enumeration of anaerobic bacterial microflora of the equine gastrointestinal tract

Samples from the duodenum, jejunum, and ileum, as well as from the cecum and colon, were obtained from 11 mature grass-fed horses. Viable counts of total culturable and proteolytic bacteria were made on habitat-simulating media containing 40% clarified ruminal fluid. The mean pHs in the duodenum, jejunum, and ileum were 6.32, 7.10, and 7.47, respectively; the mean pH decreased to 6.7 in the hindgut. The acetate concentration increased along the length of the small intestine and was the only volatile fatty acid present in this gut segment. Molar proportions of acetate, propionate, and butyrate in the hindgut were 85:10:3. Differences in bacterial counts on habitat-simulating media containing equine cecal fluid or clarified ruminal fluid were negligible. Bacterial counts showed a substantial population in the duodenum (ca. 2.9 x 10(6) per g [wet weight] of sample), and this increased to 29.0 x 10(6) in the jejunum and 38.4 x 10(6) in the ileum. Proteolytic bacteria formed a high proportion of the total culturable bacteria, especially in duodenal samples. Counts of proteolytic bacteria per gram (wet weight) of sample were 3.0 x 10(6), 15.6 x 10(6), and 22.0 x 10(6) in the duodenum, jejunum, and ileum, respectively. There was a close relationship between lumenal and mucosal bacterial counts, although actual values were lower in mucosal samples. The mucosal bacterial population in the duodenum was high relative to the lumenal population. Although the comparison of bacterial populations in the hindgut of the horse and white rhino was limited to a single animal, the results were of interest. Counts were higher in the cecum than in the colon for both the horse and the white rhino.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of anaerobic bacterial microflora of the equine gastrointestinal tract.

Samples from the duodenum, jejunum, and ileum, as well as from the cecum and colon, were obtained from 11 mature grass-fed horses. Viable counts of total culturable and proteolytic bacteria were made on habitat-simulating media containing 40% clarified ruminal fluid. The mean pHs in the duodenum, jejunum, and ileum were 6.32, 7.10, and 7.47, respectively; the mean pH decreased to 6.7 in the hindgut. The acetate concentration increased along the length of the small intestine and was the only volatile fatty acid present in this gut segment. Molar proportions of acetate, propionate, and butyrate in the hindgut were 85:10:3. Differences in bacterial counts on habitat-simulating media containing equine cecal fluid or clarified ruminal fluid were negligible. Bacterial counts showed a substantial population in the duodenum (ca. 2.9 x 10(6) per g [wet weight] of sample), and this increased to 29.0 x 10(6) in the jejunum and 38.4 x 10(6) in the ileum. Proteolytic bacteria formed a high proportion of the total culturable bacteria, especially in duodenal samples. Counts of proteolytic bacteria per gram (wet weight) of sample were 3.0 x 10(6), 15.6 x 10(6), and 22.0 x 10(6) in the duodenum, jejunum, and ileum, respectively. There was a close relationship between lumenal and mucosal bacterial counts, although actual values were lower in mucosal samples. The mucosal bacterial population in the duodenum was high relative to the lumenal population. Although the comparison of bacterial populations in the hindgut of the horse and white rhino was limited to a single animal, the results were of interest. Counts were higher in the cecum than in the colon for both the horse and the white rhino.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in rats

The effect of age and melatonin on the activity of the neuroendocrine reproductive system was studied in young cyclic (3-5 months-old), and old acyclic (23-25 month-old) female rats. Pituitary responsiveness to a bolus of GnRH (50 ng per 100 g body weight) was assessed at both reproductive stages in control and melatonin-treated (150 micrograms melatonin per 100 g body weight each day for 1 month) groups. After this experiment, female rats were treated for another month to study the influence of ageing and melatonin on the reproductive axis. Plasma LH, FSH, prolactin, oestradiol and progesterone were measured. A positive LH response to GnRH was observed in both control groups (cyclic and acyclic). However, a response of greater magnitude was observed in old acyclic rats. Melatonin treatment reduced this increased response in acyclic rats and produced a pituitary responsiveness similar to that of young cyclic rats. FSH secretion was independent of GnRH administration in all groups, indicating desynchronization between LH and FSH secretion in response to GnRH in young animals and during senescence. No effect on prolactin was observed. Significantly higher LH (3009.11 +/- 1275.08 pg ml(-1); P < 0.05) and FSH concentrations (5879.28 +/- 1631.68 pg ml(-1); P < 0.01) were seen in acyclic control rats. After melatonin treatment, LH (811.11 +/- 89.71 pg ml(-1)) and FSH concentrations (2070 +/- 301.62 pg ml(-1)) decreased to amounts similar to those observed in young cyclic rats. However, plasma concentrations of oestradiol and progesterone were not reduced. In conclusion, the results of the present study indicate that, during ageing, the effect of melatonin is exerted primarily at the hypothalamo-pituitary axis rather than on the ovary. Melatonin restored the basal concentrations of pituitary hormones and pituitary responsiveness to similar values to those observed in young rats.     

The effect of substance P (SP) infusion above the supraoptic nuclei of hypothalamus on the vasopressin content in the posterior pituitary lobe after haemorrhage in rats

Dekanski's method was used to estimate the pressor activity of the extracts of the posterior pituitary lobe in anaesthetized rats, after the infusion of 0.9% NaCl solution above the supraoptic nuclei and haemorrhage in the amount of 1.5% of body weight or after the infusion of Substance P solution above the supraoptic nuclei and haemorrhage. It has been found that the vasopressin content in the posterior pituitary lobe decreased about 20% after the infusion of 0.9% NaCl solution and haemorrhage. The infusion of Substance P above the supraoptic nuclei inhibits the loss of vasopressin from the pituitary caused by haemorrhage.     

Regional Distribution of Prostaglandins D2, E2, and F2α and Related Enzymes in Postmortem Human Brain

Abstract: The presence of prostaglandins D₂, E₂, and F_2α was demonstrated and their contents measured in various regions of postmortem human brain, pineal body, and pituitary by using specific radioimmunoassays and gas chromatography-mass spectrometry. The three prostaglandins were widely distributed in similar concentrations ranging from several hundred pg/g wet weight to about 40 ng/g wet weight. Prostaglandins D₂ and E₂ showed consistent and similar regional distributions in all six brains tested; amounts were high in pineal body, pituitary, olfactory bulb, and hypothalamus. On the other hand, prostaglandin F_2α was distributed more evenly. Prosta- glandin D synthetase and prostaglandin E synthetase activities were found in cerebrum homogenate from a single subject and were recovered from the 100,000 × g supernatant. The presence of 1 m M glutathione, reduced form, markedly stimulated the activity of prostaglandin E synthetase, but did not affect prostaglandin D synthetase activity. Activity of 15-hydroxyprostaglandin dehydrogenase was found in the cerebrum homogenate and was partially purified. This enzyme required NADP as a cofactor and copurified with prostaglandin E 9-ketoreductase.