ABSTRACT
The modification of canine fibrinogen with citraconic anhydride modified the epsilon-amino groups of the fibrinogen and at the same time generated additional negative charges into the protein. The addition of thrombin to the modified fibrinogen did not induce polymerization; however, the fibrinopeptide was released at a faster rate than from the unmodified fibrinogen. The physical properties of the citraconylated fibrinogen were markedly altered by the modification of 50-60 lysine residues in one hour. A modified fibrinopeptide-A was released by thrombin from the modified fibrinogen and was electrophoretically more anionic than the unmodified fibrinopeptide-A. Edman analysis confirmed the modification of the lysine residue present in the peptide. The rate of removal of citraconylated fibrinopeptide-A from modified fibrinogen by thrombin was 30 to 40 percent greater than the cleavage of unmodified fibrinopeptide-A from unmodified fibrinogen. However, the modification of 60 or more lysine residues in the fibrinogen produced a decrease in the rate of cleavage of citraconylated fibrinopeptide-A. The results suggest that additional negative charge in the vicinity of the attachment of fibrinopeptide-A to canine fibrinogen aids in the removal of the peptide by thrombin.
Subject(s)
Citraconic Anhydrides/pharmacology , Fibrinogen/metabolism , Furans/pharmacology , Thrombin/pharmacology , Amino Acids/analysis , Animals , Chemical Phenomena , Chemistry , Dogs , Fibrinopeptide A/analysis , Fibrinopeptide A/metabolism , Kinetics , Lysine/analysis , Lysine/metabolism , Macromolecular SubstancesABSTRACT
The trimethyl oxonium ion specifically modified the free carboxyl groups of fibrinogen. This esterification process resulted in the polymerization of the modified fibrinogen molecule with the production of a polymeric material that resembled the physiologically formed fibrin clot. The extent of methylation of fibrinogen was evaluated by methoxyl determination at each step of the polymerization process. The modified fibrinogen polymerized in approximately ten min with a minimum number of methyl groups being incorporated into the fibrinogen molecule. In this manner, it was shown that modification of carboxyl groups in the fibrinogen by a group-specific methylating agent results in polymerization of fibrinogen. The sites of methylation were ascertained by chromatographic analysis which resulted in the identification of beta-methyl aspartic acid and gamma-methyl glutamic acid derivatives of the fibrinogen. The analytical methods applied were not able to detect the methylation of any additional amino acid residues in the polymerized-methylated fibrinogen. Based on this experimental data, it was formulated that the methylation of fibrinogen involved the esterification of the carboxyl groups of aspartic and glutamic acid with the resultant reduction of negative repulsion between the fibrinogen molecules and thereby culminated in the polymerization of the modified fibrinogen.
Subject(s)
Fibrinogen , Onium Compounds/pharmacology , Animals , Aspartic Acid , Carboxylic Acids , Cattle , Chemical Phenomena , Chemistry, Physical , Esterification , Glutamates , Glutamic Acid , Macromolecular Substances , MethylationABSTRACT
Neurotensin (NT), administered intracisternally to mice, produced significant dose-dependent antinociception in three analgesic tests: tail immersion, hot-plate and acetic acid writhing. Naloxone (1-5 mg/kg), an opiate antagonist administered i.p. 20 min before NT administration, did not significantly alter NT-induced antinociception in any of these tests; naloxone did significantly reverse beta-endorphin-induced antinociception. However, centrally and peripherally administered thyrotropin-releasing hormone antagonized NT-induced (but not beta-endorphin-induced) antinociception. Equimolar doses of another tripeptide (Pro-Leu-Gly-NH2; melanostatin) did not alter the effects of NT. The data obtained in this study confirm NT-induced antinociception, provide further evidence that NT does not activate naloxone-sensitive opiate receptors and demonstrate that this brain effect of NT is antagonized by thyrotropin-releasing hormone. These findings therefore support the hypothesis that NT and thyrotropin-releasing hormone are functional antagonists in the central nervous system.
Subject(s)
Analgesics , Neurotensin/pharmacology , Thyrotropin-Releasing Hormone/pharmacology , Animals , Body Temperature/drug effects , Dose-Response Relationship, Drug , MSH Release-Inhibiting Hormone/pharmacology , Male , Mice , Naloxone/pharmacology , Neurotensin/antagonists & inhibitors , Reaction Time/drug effectsABSTRACT
Polymerization was accomplished in the fibrinogen system by methylation with diazomethane, thionyl chloride and dimethyl sulphate. The duration and extent of polymerization were dependent on the modifying agent which was applied to the fibrinogen system. When fibrinogen was methylated with a very narrow range group-specific methylating agent, like dimethyl sulphate, the polymerization process was accelerated and proceeded with a reduction in the extent of modification of that obtained with the other methylating reagents utilized in these experiments. Chromatographic analysis revealed that diazomethane and thionyl chloride induced both O-methylation and N-methylation, as well as esterification of the carboxylate groups of aspartic and glutamic acid in fibrinogen. However, dimethyl sulphate resulted primarily in esterification accompanied by a small amount of histidine methylation. The proposed mechanism for the polymerization is through the esterification of the aspartic and glutamic acid residues which produces an increased protein-protein interaction resulting in polymer formation.
Subject(s)
Fibrinogen/analogs & derivatives , Amino Acids , Biopolymers , Chromatography , Diazomethane , Fibrinogen/isolation & purification , Methylation , Peptide Fragments/analysis , Sulfates , ThionesABSTRACT
Neurotensin (NT), an endogenous tridecapeptide, produces significant hypothermia after intracisternal (i.c.) or intracerebroventricular (i.c.v.) administration in microgram quantities in a variety of laboratory animals. The present study sought to clarify the mechanism of the hypothermic action by utilizing pharmacological treatments which alter the function of brain neurotransmitter systems. Pretreatment of rats with anti-muscarinic (atropine), anti-noradrenergic (propranolol, a beta-blocker; phenoxybenzamine, an alpha-blocker) or anti-opiate (naloxone) agents did not significantly alter NT-induced hypothermia. Similarly depletion of brain serotonin (5-HT) with parachlorophenylalanine did not affect NT-induced hypothermia. However, depletion of brain catecholamine content with 6-hydroxydopamine resulted in a significant potentiation of NT-induced hypothermia as did pretreatment with haloperidol, a dopamine (DA) receptor antagonist. Furthermore, in rats with selective depletions of brain DA, but not norepinephrine (NE), NT-induced hypothermia was significantly augmented. Thus an interaction between brain DA systems and NT appears likely. These data indicate that NT-induced hypothermia is not dependent on intact functional activity of NE, 5-HT, muscarinic ACh or endogenous opiate systems but suggests interactions between brain DA circuits and NT. In other experiments, NT-induced hypothermia was found to be antagonized significantly by i.c. injection of thyrotropin-releasing hormone (TRH), but not by pretreatment with L-triiodothyronine. Another endogenous tripeptide (Pro--Leu--Gly--NH2, MIF-I) had no effect. Thyroidectomy (THX) significantly potentiated NT-induced hypothermia; NT administered i.c. significantly reduced the high serum TSH levels of THX rats. Thus, NT and TRH, two endogenous peptides, appear to be antagonists in certain systems.
Subject(s)
Body Temperature Regulation/drug effects , Hypothalamo-Hypophyseal System/drug effects , Neurotensin/pharmacology , Receptors, Dopamine/drug effects , Thyroid Gland/drug effects , Animals , Fenclonine/pharmacology , Haloperidol/pharmacology , Hydroxydopamines/pharmacology , Male , Rats , Thyrotropin/pharmacology , Thyrotropin-Releasing Hormone/pharmacology , Triiodothyronine/pharmacologyABSTRACT
To study the effect of the acidic amino acid residues on the physiological polymerization and clot formation of fibrinogen, the fibrinogen system was polymerized by interaction with diazomethane, a specific group reagent which modifies the carboxylic acid residues via the process of methylation. The extent of methylation of fibrinogen by diazomethane was estimated by methoxyl determination on the modified fibrinogen. Under the present experimental conditions, no significant amounts of ammonia were detected as a result of amide hydrolysis concomitant with esterification of the fibrinogen. Chemical methylation of approximately 214 residues resulted in polymerization of the fibrinogen molecule to a product which resembled the physiological clot formation. The application of paper chromatographic techniques identified the modification of other amino acid residues in addition to the methylation of the carboxylic acid groups of glutamic and aspartic acids. These results are interpreted in terms of methylation of carboxylic acid groups in fibrinogen by diazomethane providing a reduction of both negative charge and intermolecular repulsion, thereby enabling the modified fibrinogen molecules to polymerize.
Subject(s)
Diazomethane , Fibrinogen/analogs & derivatives , Animals , Biopolymers , Cattle , MethylationSubject(s)
Body Temperature Regulation , Neurotensin/physiology , Animals , Anura , Cold Temperature , Columbidae , Cricetinae , Fishes , Gerbillinae , Guinea Pigs , Haplorhini , Lizards , Marmota , Mesocricetus , Mice , Neurotensin/metabolism , Rabbits , Rats , Sciuridae , Species SpecificityABSTRACT
The antinociceptive and hypothermic effects of intracisternal administration of 11 endogenous neuropeptides and morphine were evaluated in mice. Of the substances tested, only neurotensin (NT) and beta-endorphin exerted significant antinociceptive and hypothermic effects; NT was the most potent in inducing hypothermia whereas beta-endorphin was the most potent antinociceptive agent via this route of administration. Both NT, and beta-endorphin were, on a molar basis, considerably more potent antinociceptive agents than morphine, [Met]enkephalin, or [Leu]enkephalin. NT-induced analgesia and hypothermia both were significantly dose-dependent. Substance P was found to produce significant hyperalgesia and hyperthermia. Bombesin produced a significant hypothermic effect, whereas somatostatin and luteinizing hormone-releasing hormone (luliberin) produced hyperthermia. None of the other peptides studies [bradykinin, thyrotropin-releasing factor (thyroliberin), melanocyte-stimulating hormone release-inhibiting factor (melanostatin), somatostatin, [Met]enkephalin, and [Leu]enkephalin] produced any significant alterations in colonic temperature or response to a noxious stimulus with the doses tested. These data demonstrate that NT and beta-endorphin, two endogenous brain peptides, are potent in inducing hypothermia and in producing an antinociceptive state.
Subject(s)
Body Temperature Regulation/drug effects , Endorphins/pharmacology , Enkephalins/pharmacology , Morphine/pharmacology , Neurotensin/pharmacology , Pain , Peptides/pharmacology , Animals , Body Temperature/drug effects , Dose-Response Relationship, Drug , Hormones/pharmacology , Male , Mice , Structure-Activity RelationshipABSTRACT
Intracisternal (IC) neurotensin (NT) produces muscle relaxation in the Julou-Courvoisier traction test, a screening procedure utilized for assessing neuroleptic drug activity. A dose-response relationship was not observed. IC administration of thyrotropin-releasing hormone (TRH) totally abolished the effects of NT. Two other peptides, substance P and bradykinin, were inactive in the traction test. These data provide further evidence for CNS effects on NT and indicate that this peptide exerts neuroleptic-like activity in a screening test for antipsychotic agents.
Subject(s)
Muscle Contraction/drug effects , Muscle Relaxation/drug effects , Neurotensin/pharmacology , Animals , Bradykinin/pharmacology , Cisterna Magna , In Vitro Techniques , Injections , Male , Mice , Neurotensin/administration & dosage , Substance P/pharmacology , Thyrotropin-Releasing Hormone/pharmacology , Time FactorsABSTRACT
Peripheral administration of the COOH-terminal octapeptide of cholecystokinin in doses from 1 to 100 micrograms per kilogram of body weight (0.25 to 25.0 micrograms per rat) significantly antagonized tail pinch-induced eating in rats, an animal model for stress-induced human hyperphagia. Centrally administered cholecystokinin was effective only in high doses (3 micrograms into the cerebral ventricle). The finding that the minimal effective dose of cholecystokinin in suppressing stress-induced appetitive behavior is smaller after peripheral than central administration suggests that the peptide is acting on peripheral, as opposed to central nervous system, substrates.
Subject(s)
Cholecystokinin/pharmacology , Feeding Behavior/drug effects , Animals , Behavior, Animal/drug effects , Bradykinin/pharmacology , Cholecystokinin/administration & dosage , Dose-Response Relationship, Drug , Humans , Male , Peptide Fragments/pharmacology , Rats , Stress, PsychologicalABSTRACT
Canine peptide-A was demonstrated to be an active vasopressor substance. The effect of intravenous injection of canine peptide-A was a prolonged rhythmic vasoconstriction and is unlike any previously described vasopressor peptides. The intravenous administration of canine peptide-A resulted in a maximum increase in mean blood pressure of 19.0 mm of mercury at a dose level of 2.5-10-minus 2 mumol. This pressor effect exhibited a log proportionality to the injected dose of peptide. The duration of the vasopressor effect was 30 min or greater. A rhythmic variation in pressor response was also observed in all experiments. The canine AP and B peptides were inactive when tested under the same conditions. The relationship between structure and biological activity was investigated. Structural degradation of half of the peptide molecule was performed without loss of vasopressor activity. The canine peptide-A is postulated as assisting in local hemostasis by vasoconstriction.
Subject(s)
Blood Pressure/drug effects , Fibrinogen/pharmacology , Peptide Fragments/pharmacology , Amino Acids/analysis , Animals , Biological Assay , Chymotrypsin , Dogs , Dose-Response Relationship, Drug , Peptide Fragments/isolation & purification , Rats , Structure-Activity Relationship , Time FactorsABSTRACT
Bovine peptide-B from fibrinogen was capable of accelerating the structural and enzymatic effects associated with superprecipitation of myosin B. The rate of superprecipitation coupled with the hydrolysis of ATP are increased during the structural transformation. In the concentration range from 10-8 to 10-4 M peptide-B, the rate of superprecipitation is increased 12-fold while the hydrolysis of ATP doubles and the time to reach the final extent of superprecipitation is decreased 68%. Under these same conditions, the hydrolysis of ATP by myosin A was unaffected. The concentrations of magnesium and calcium were between 10 and 20 muM, and no additional divalent metal ions were added to the system. Superprecipitation was treated as a model for muscle contraction to explain the in vivo studies of bovine peptide-B in which the peptide behaves as a vasopressor substance producing vascular vasoconstriction. A possible mechanism for the participation of bovine peptide-B in the model for muscle contraction, based on the polarizing interaction of the highly charged density of negativity of the peptide with the actomyosin complex, is presented. Furthermore, bovine peptide-B is speculated as participating in vasoconstriction via attachment to some smooth muscle receptor.
Subject(s)
Actomyosin/metabolism , Adenosine Triphosphatases/metabolism , Fibrinogen/analysis , Muscle Contraction/drug effects , Muscles/metabolism , Peptides/pharmacology , Vasoconstrictor Agents/pharmacology , Adenosine Triphosphate/metabolism , Animals , Calcium/metabolism , Cattle , Hydrolysis , In Vitro Techniques , Magnesium/metabolism , Models, Biological , Muscles/enzymology , Myosins/metabolism , SpectrophotometryABSTRACT
Bovine peptide-B from fibrinogen was active in the hemostasis of rat tail arterioles. The time of bleeding exhibits an inverse log proportionality to the concentration of bovine peptide-B. The peptide produced a 10-fold decrease in the time of bleeding at the highest concentration tested. Prolonged incubation of the peptide with the system was unnecessary and it appeared to participate immediately as a hemostatic agent. These findings suggest that hemostasis was due to vasoconstriction since coagulation time remained constant as the bleeding time decreased with increasing concentration of peptide-B. Bovine peptide-B is interpreted as a physiological substance which probably acts on some smooth muscle receptor.