Blood pressure responses of conscious rats to intravenous administration of enkephalin derivatives (D-ala methionine and leucine enkephalinamide, and methionine and leucine enkephalinamide)

The present study was designed to determine the blood pressure (BP) responses of conscious rats given intravenous (IV) injections of enkephalin derivatives (D-ala²-methionine enkephalinamide, DAMEA; D-ala²-leucine enkephalinamide, DALEA; methionine enkephalinamide, MEA; leucine enkephalinamide, LEA) and the receptor mechanisms mediating the resultant change in BP. IV injection of 1.6–16.0 nmoles of DAMEA or DALEA caused a transient but potent decrease in mean arterial pressure (MAP) and mean heart rate (MHR). LEA and MEA (16.0 nmoles) given IV produced slight pressor responses, which were not associated with concomitant tachycardia whereas 48 nmoles of MEA elicited a hypotensive effect accompanied by a fall in MHR. Pretreatment studies whereby various receptor antagonists (naloxone, diprenorphine, phentolamine, D-L-propranolol or atropine) were given IV 5 min before subsequent IV administration of DAMEA, DALEA, MEA or LEA (16 nmoles) showed that naloxone, diprenorphine and atropine blocked the depressor and bradycardic effects of DALEA and DAMEA. Naloxone and phentolamine suppressed the pressor reponse of both MEA and LEA (16.0 nmoles) while diprenorphine blocked the rise in MAP to only MEA. The results show that DAMEA and DALEA mediate their depressor actions in conscious rats via a negative chronotropic effect through an interaction of muscarinic cholinergic receptors on the myocardium. It is suggested that the pressor response of MEA and LEA may be produced via an -receptor mediated effect on the peripheral vasculature to cause vasoconstriction.     

Pressor effects of systemic administration of methionine and leucine enkephalin in the conscious rat

Experiments were designed using conscious Sprague-Dawley rats to determine the blood pressure (BP) and heart rate (HR) responses to intravenous doses of (1) the adrenal catecholamines noradrenaline (NA) and adrenaline (A), (2) adrenal pentapeptides methionine enkephalin (ME) and leucine enkephalin (LE), (3) combination (i.v.) injections of both ME or LE with NA or A that modulate the hemodynamic responses when the adrenal catecholamines were given alone, and (4) the possible receptor mechanisms mediating the resultant BP and HR response to i.v. pentapeptide administration. NA (0.48 and 2.4 nmol) and A (0.3 and 1.5 nmol) given i.v. evoked potent, dose-related pressor responses associated with reflex bradycardia. ME and LE (1.6 - 48 nmol) elicited transient (10-20 s) increases in mean arterial pressure (MAP), which was associated either with no change in mean heart rate (MHR), such as ME, or with slight bradycardia (i.e., LE). Combining ME or LE (16 nmol) with NA (2.4 nmol) or A (0.3 or 1.5 nmol) did not change MAP and MHR from when these respective doses of NA or A were given alone. However, 16 nmol of ME or LE with a low dose of NA (0.48 nmol) increased the pressor response compared with NA (0.48 nmol) given alone. Other experiments whereby specific receptor blockers (naloxone, diprenorphine, atropine, propranolol, phentolamine or guanethidine) were given i.v. 5 min before subsequent i.v. administration of LE or ME (16 nmol) indicated that only phentolamine or guanethidine could completely suppress the pressor responses of LE and ME. Naloxone and diprenorphine pretreatment attenuated the pressor response of LE but did not affect the BP response to ME.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in brain enkephalin immunostaining after acute carbon disulfide exposure in rats

Carbon disulfide neurotoxic mechanism in the brain is still not completely clear. In this work, the effect of carbon disulfide exposure in rats on the enkephalinergic neuromodulatory system is described. Caudatus-putamen showed no changes in immunostaining for met-enkephalin when compared with controls. However, a marked reduction in met-enkephalin immunostaining in the central amygdaloid nuclei and the globus pallidus was measured, with a parallel elevation in the lateral septal nucleus and the parietal cortex. It is suggested that enkephalinergic neuromodulatory system could play a role in carbon disulfide neurotoxicity.     

Comparison of synenkephalin and methionine enkephalin immunocytochemistry in rat brain

Immunocytochemistry using an antiserum to the C-terminal octapeptide of synenkephalin, proenkephalin(63–70), was performed throughout the rat brain and revealed numerous immunopositive fibers and some cell bodies. The morphology and distribution of synenkephalin immunoreactivity was extremely similar to that of a commercial methionine enkephalin (Met-ENK) antiserum. Colchicine pretreatment allowed the immunostaining of cell bodies not otherwise possible without pretreatment, but did not affect the distribution of immunoreactive fibers. Using 6 μm serial sections, we were able to colocalize synenkephalin and Met-ENK immunoreactivities in gigantocellular neurons of the medullary reticular formation. Preabsorption of the antiserum with [Tyr⁶³]proenkephalin(63–70) octapeptide (YEESHLLA) completely eliminated immunoreactivity in the rat brain, while preabsorption with all other peptides used had no detectable effect. We conclude that our antiserum to synenkephalin is specific for enkephalinergic cell bodies, fibers and terminals. The synenkephalin antiserum used in these studies may have advantages over other antisera utilized for immunocytochemical detection of proenkephalin gene expression.     

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin.

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. N alpha-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of alpha-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin- and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.     

D-amphetamine-induced hypothermia and hypermotility in rats: Changes after systemic administration of beta-endorphin

Systemically administered beta-endorphin was tested in rats for its ability to modify the hypothermia and hypermotility induced by d-amphetamine. Colonic temperature and motor activity were measured in a cold (4°C) ambient temperature in animals given IP injections of beta-endorphin (0.1, 1.0, or 3.0 mg/kg), naloxone (10 mg/kg), or morphine (30 mg/kg). The same measurements were taken in animals given beta-endorphin (1.0 mg/kg) in combination with naloxone or saline pretreatment and d-amphetamine (15 mg/kg) or saline post-treatment. Morphine alone had a biphasic effect on thermoregulation, but did not affect d-amphetamine-induced hypothermia. Activity scores were decreased by morphine, in both d-amphetamine and saline treated animals. The thermal response of rats to beta-endorphin alone was variable, depending on dosage, but all 3 dosages partially blocked the hypothermic effect of d-amphetamine. Naloxone blocked the thermal effects of both beta-endorphin and d-amphetamine. Motor activity tended to be decreased by naloxone, regardless of amphetamine treatment, but beta-endorphin tended to increase activity in amphetamine-treated animals and reduce it in saline-treated controls. In their actions on both thermoregulation and activity, naloxone and beta-endorphin appeared to interact independently with d-amphetamine, often producing effects in the same direction, but in combination, they tended to be mutually inhibitory.     

Plasma uptake and in vivo metabolism of [Leu]enkephalin following its intraperitoneal administration to rats

To understand better how [Leu]enkephalin (LE) acts to modulate learning and memory in rats, the plasma uptake, disappearance, and metabolism of LE were investigated following its intraperitoneal administration. Concentrations of [3H]-LE and its radioactive metabolites were determined by thin layer chromatography in plasma samples withdrawn from rats at various times after injection of peptide. As measured in rats receiving an IP injection of a dose of LE (3 micrograms/kg) that impairs active avoidance conditioning, the LE was very rapidly metabolized, with greater than 95% of plasma [3H] in the form of metabolites by 1 min after injection. Despite this rapid metabolism, low but measurable quantities of intact LE were detectable in plasma at all sampling times. Consistent with a greater potency of D-Ala2-[D-Leu5]enkephalin (DADLE) than of LE in modulating avoidance conditioning, DADLE was less rapidly metabolized than was LE following its IP administration. The metabolism of DADLE and LE in vivo was more rapid than it was in plasma in vitro, suggesting a role for membrane bound enzymes in the metabolism of IP-administered enkephalins. The data demonstrate that, despite a rapid hydrolysis of LE in vivo, sufficient LE is present in plasma following IP administration of a behaviorally active dose to support a role of circulating intact LE in the modulation of avoidance conditioning.     

Psychostimulation of Wistar rats after intraperitoneal administration of high-molecular heparin

The influence of repeated administration of heparin in learning was studied in Wistar rats (n = 20). High-molecular heparin (Serva, Germany, 10 kDa, activity of 180 IU/mg) was intraperitoneally injected daily in the dose of 64 IU/kg in a volume of 0.3 ml during 5 days prior to 4-link freechoice operant conditioning in a complex maze. As distinct from 40% of control rats, practically all heparin-treated rats were successful in conditioning. Parameters such as rate and efficiency of learning, behavior organization, habit stability, and extinction of errors were significantly better than in control animals. Anxiety was decreased, but sensitivity to external factors was increased in comparison with the control rats. The data obtained suggest that prior treatment with high-molecular heparin has a strong psychostimulant effect through activation of biogenic amines and formation of complexes with them.     

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro

 Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i. p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage- colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i. p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth. Received: 11 April 1996 / Accepted: 21 May 1996     

Development of a capillary zone electrophoresis assay to examine the disposition of [d-pen]enkephalin in rats

A novel capillary zone electrophoresis (CZE) assay method was developed to evaluate the systemic disposition of [d-pen^2,5]enkephalin (DPDPE) in rats. DPDPE was recovered from serum samples (200 μl) by solid-phase extraction. Complete resolution of DPDPE and the internal standard ([d-ser²]leucine-enkephalin; DSLET) from other serum components was achieved within 15 min on a 50-μm I.D. capillary column with borate buffer (25 mM, pH 8.3). The peak-height ratio (DPDPE to DSLET) was linear through 100 μg/ml, with a detection limit of 250 ng/ml in serum, when absorbance of the column eluent was monitored at 210 nm. Serum samples obtained from rats after a 10 mg/kg intravenous bolus dose of DPDPE were analyzed with the present CZE method. The results suggest that CZE is a useful technique for quantitating therapeutic peptides in biological matrices.     

Metabolism of glycosaminoglycans in rats during methionine deficiency and administration of excess methionine

Methionine deficiency in rats caused significant decrease in the concentration of many sulphated glycosaminoglycans in the aorta and other tissues, while administration of excess methionine caused an increase in these constituents. The activity of some important biosynthetic enzymes decreased in methionine deficiency and increased on administration of excess methionine. No uniform pattern was observed in the changes in the activity of enzymes concerned with degradation of glycosaminoglycans. The concentration of 3′-phosphoade-nosine 5′-phosphosulphate and the activities of the sulphate activating system and sulpho-transferase were decreased in methionine deficiency, while feeding excess methionine did not affect these parameters as compared to controls.     

吗啡对猕猴瞳孔光反应能力的影响

光照能明显改变正常人和动物瞳孔的大小,而精神疾病及药物滥用则影响人和动物瞳孔对光的反应性.因此,瞳孔对光反应异常可以用作检测精神疾病和药物滥用的指标.有关药物滥用是如何影响瞳孔对光的反应性的研究还很少.为定量地测量成瘾性药物对瞳孔光反应变化的影响,该文采用猕猴为实验对象,通过在黑暗环境中测量猕猴在吗啡给予前和吗啡给予后的不同时间段,其瞳孔直径大小以及其对光反应能力的变化情况,来系统研究吗啡是如何影响这种非自主性反射系统的.研究发现,吗啡给予降低了猕猴在黑暗环境中的扩瞳反应,并且降低了瞳孔对光反应的收缩率.该文为将瞳孔对光反应特征用作鉴定吸毒者的检测手段提供了实验依据.     

Analgesic effects of enkephalin analogs in rats

The analgesic effects of intracerebroventricular injections of Met-enkelphalin and five of its analogs in a dose of 10 μg each were quantified with a hot plate test in rats. Two analogs showed analgesic effect. ?D-Ala₂, Met⁵⊥-enkephalinamide and short-lasting analgesic effect. ?D-Ala², Met⁵⊥-enkelphalinamide had a weak (DALA) had a striking and long-lasting analgesic effect. However, sulfation of tyrosine residue totally abolished the analgesic action of DALA. The analgesic effect of DALA was not affected by preinjection of its sulfated analog.     

Circadian Rhythm of the Pupillary Response to Morphine and to Naloxone

Mice respond to morphine with characteristic mydriasis which is antagonized by naloxone. The present study presents data on the diurnal variation of these responses. The mydriatic response is at its highest level at 0800 and has a nadir at 2400. This effect is not influenced by ambient light conditions. The miotic response to naloxone in the morphine-dilated pupil is maximal between 1200 and 1600 and minimal at 0400. The curves of the responses to the two drugs, therefore, differ as do their acrophases. Pharmacokinetic factors are probably not responsible for the observed variations. It is speculated that these diurnal changes may reflect cyclic alteration in the affinity of the opiate receptors to agonists and antagonists, respectively, or in the number of available receptors.     

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[³H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.     

Possible autocrine enkephalin regulation of catecholamine release in human pheochromocytoma cells

AIMS: Pheochromocytomas are catecholamine-secreting tumors that also synthesize and secrete several neuropeptides, including opioids. A negative regulation of catecholamine secretion by opioids has been postulated in chromaffin cells. However, results obtained so far are contradictory when referred to human pheochromocytomas. The aim of this study was to define the role of locally produced enkephalins on catecholamine release in human pheochromocytoma cells. MAIN METHODS: Cells obtained from eleven human pheochromocytomas of different genetic origins were cultured for 5 days. Cultures were maintained under basal condition or under enkephalin, dexamethasone and naloxone alone or in combination with enkephalin or dexamethasone-stimulated conditions. Catecholamine and enkephalin levels in the culture medium were measured by HPLC-ED and RIA respectively. KEY FINDINGS: Enkephalin induced a decrease in norepinephrine levels in all tumor cultures. Dexamethasone treatment, which increased enkephalin levels, also decreased catecholamine levels. On the other hand, the addition of naloxone to the cultures reverted to normal the inhibitory action exerted by enkephalin and dexamethasone treatments. SIGNIFICANCE: These results suggest the existence of an autocrine negative regulatory loop exerted by enkephalin on norepinephrine release in human pheochromocytoma cells.     

Shigellosis murine model established by intraperitoneal and intranasal route of administration: a comparative comprehension overview

Shigellosis, a major cause of mortality and morbidity, requires development of effective intervention strategy for which animal model mimicking human pathology is essential. Among various animal models for shigellosis, mice being more convenient have been used wherein intraperitoneal and intranasal routes are preferred. With the aim to comprehend the comparative pathophysiological indicators, we have examined relatively high and low dose of Shigella flexneri administered through intraperitoneal and intranasal routes in mice. Characterization of these two models along with the resulting pathophysiology of shigellosis adds to our understanding and offers suitable models appropriate to the objectives of the study.