Difference in receptor-binding contributes to difference in biological activity between the unique guinea pig GnRH and mammalian GnRH

In the current study, we compared the in vitro potency of a unique form of gonadotropin-releasing hormone (GnRH) present in the brain of the guinea pig (gpGnRH) with that of mammalian GnRH (mGnRH) as well as their binding affinities to the GnRH receptor. In gpGnRH, the highly conserved histidine in position 2 (His²) and leucine in position 7 (Leu⁷) are substituted by tyrosine and valine, respectively. In vivo, gpGnRH was shown to be less potent than mGnRH, possibly in part because of higher susceptibility to enzymatic degradation. In the present in vitro experiments, we observed that gpGnRH was less potent than mGnRH in stimulating the release of luteinizing hormone (LH) from primary pituitary cell cultures of the rat, and at lower concentrations from primary pituitary cell culture of the guinea pig, too. These results were confirmed by radioligand-binding studies. It is concluded that the lower biological activity of gpGnRH in both rat and guinea pig may be explained by the difference in binding to the target cells, although additional factors such as proteolytic degradation may also contribute to the observed phenomenon.     

Genotoxicity of the cooked-food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food-borne mutagens 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p less than 0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species-dependent.     

GnRH 相关肽在啮齿类前脑及下丘脑分布的免疫组织化学研究

人和大鼠的促性腺激素释放激素原(ProGnRH),由 GnRH 和 GnRH 相关肽(GAP)组成。本文应用3种分别抗人和大鼠 GAP N-末端、中段和 C-末端的抗血清和 ABC 免疫酶法,对大鼠、小鼠和豚鼠脑 GAP 神经元进行了观察。3种动物的 GAP 神经元分布相似,GAP 神经元胞体主要存在于隔-视前区,以终板血管器附近的斜角带最为集中。视上核附近亦有较多的 GAP神经元。GAP 纤维广泛存在于前脑和下丘脑,终止于终板血管器和正中隆起。3种 GAP 抗血清中,以抗 N-末端者染出的免疫反应阳性成分多,染色深。GAP 神经元与相邻切片上所染的 GnRH神经元的胞体、纤维和终末的形态和分布相同。本文的观察和其他有关的研究提示,哺乳类具有相同的 ProGnRH,其加工产物 GAP(或其裂解片段)与 GnRH 共同分泌到垂体门脉,调节垂体前叶的激素分泌。     

Immunocytochemical localization of mammalian GnRH (gonadotropin-releasing hormone) and chicken GnRH-II in the brain of the European silver eel (Anguilla anguilla L.)

Using specific antibodies for the two molecular forms of gonadotropin-releasing hormone (GnRH) present in the European eel, Anguilla anguilla, (mammalian GnRH, mGnRH, and chicken GnRH II, cGnRH-II), we employed immunocytochemistry to determine the distribution of these two peptides in the brain and in the pituitary. The results indicate that mGnRH and cGnRH-II are localized in different neurons: mGnRH-immunoreactive (ir) perikaria were observed in the olfactory bulbs, the junction between olfactory bulbs and telencephalon (nucleus olfactoretinalis), the telencephalon, the preoptic region and the mediobasal hypothalamus. These cell bodies are located along a continuum of ir-fibers that could be traced from the olfactory nerve to the pituitary. Mammalian GnRH-ir fibers were detected in many parts of the brain (olfactory bulbs, ventral telencephalon, hypothalamus, optic tectum, mesencephalon) and in the pituitary. Chicken GnRH-II-ir cell bodies were detected in the nucleus of the medial longitudinal fasciculus of the midbrain tegmentum, but only scattered fibers could be detected in different parts of the brain. The pituitary exhibited very few cGnRH-II-ir fibers, contrasting with an extensive mGnRH innervation. These results are in agreement with our previous data obtained in the same species using specific radioimmunoassays for mGnRH and cGnRH-II. They demonstrate a differential distribution of the two forms of GnRH in the brain of the eel, as in the brain of some other vertebrate species, and suggest differential physiological roles for the two GnRH forms in the eel. They also provide information concerning the evolution of the GnRH systems in vertebrates.     

Evaluation of the C-terminal C5a effector site with short synthetic C5a analog peptides

Biological activities have been determined for a series of 18 peptides based on the C-terminal sequence of human or rat C5a. Lysosomal enzyme release was tested in two cell types, the promyelotic leukemia cell line U937 and polymorphonuclear leukocytes. In addition, an ATP-release assay with guinea pig platelets was performed. It was demonstrated that the C-terminal octapeptide 67-74 of human C5a represents the minimal sequence required to induce a measurable biological signal in all assays. Extending this peptide to a length of 21 amino acids produced at best only a slight enhancement of potency. Amino acid replacements with either tryptophanyl or phenylalanyl residues in positions between 65–69 either increased potency (at position 67), or abrogated potency (at position 66) in the two lysosomal enzyme assays. N-terminal acylation with the fluorenylmethoxy-carbonyl-aminohexanoyl group slightly enhanced C5a potency. In desensitization experiments with guinea pig platelets all peptides with a C5a activity were able to desensitize not only the C5a but also the C3a responses.     

Histamine- and serotonin-releasing activity of the supernatants from guinea pig spleen cell cultures

The action of supernatants from cultivated in vitro guinea pig spleen cells on the mast cells of guinea pigs, rats and hamsters was studied. It was found that supernatants from guinea pig spleen cell cultures are potent to release histamine from mast cells of the examined populations in a dose-dependent fashion. Histamine release from heterologous mast cells (especially from rat pleural mast cells) was significantly higher than that from homologous mesenteric mast cells. It was also demonstrated (on rat mast cells) that guinea pig spleen cell supernatants possessed not only histamine but 5-hydroxytryptamine releasing activity as well. Rat pleural mast cells were more sensitive and released more serotonin after challenge with spleen cell supernatants than peritoneal cells.     

Pineal serotonin-N-acetyltransferase activity in four mammalian species

The activity of pineal serotonin-N-acetyltransferase (NAT) was compared in four mammalian species: the albino rat, the golden hamster, the Mongolian gerbil and the English short-hair guinea pig. In all species a night-time elevation in pineal NAT activity was apparent. In the rat, pineal NAT activity exhibited a dark:light ratio of 60:1; in the hamster and gerbil the ratio was 3:1 while in the guinea pig the dark:light ratio was 1.5:1. With the exception of the guinea pig, the greatest pineal NAT activity was recorded at 04.00 h (8 h after onset of darkness). In the guinea pig the maximal NAT activity was attained at 24.00 h (4 h after onset of darkness).     

An investigation of the effects of maternal separation and novelty on central mechanisms mediating pituitary-adrenal activity in infant guinea pigs (Cavia porcellus)

In mammalian species in which the young exhibit a strong filial attachment (e.g., monkeys, guinea pigs), numerous studies have shown that even brief separation from the attachment figure potently elevates circulating concentrations of glucocorticoids and adrenocorticotropic hormone (ACTH). However, effects of separation on central regulation of this stress response are not known. Therefore, we investigated central mechanisms mediating pituitary-adrenal activation during maternal separation and novelty exposure in guinea pig (Cavia porcellus) pups. Corticotropin-releasing factor (CRF) mRNA expression in the hypothalamic paraventricular nucleus (PVN), and plasma cortisol and ACTH levels, were elevated only during separation in a novel environment. C-Fos activity was elevated in the medial amygdala (MeA) and reduced in the bed nucleus of the stria terminalis (BNST) during novelty exposure, regardless of separation. On the other hand, c-Fos activity was elevated in the PVN during separation, regardless of novelty exposure. These results demonstrate independent and combined effects of separation and novelty in regions of the guinea pig CNS that regulate pituitary-adrenal activity. Moreover, they suggest that a pathway from MeA to BNST to PVN mediates responses to novelty in the guinea pig pup, as in the adult rat, though inputs from other cell populations appear required to fully account for the HPA activity observed here.     

Production, assay and partial characterization of guinea pig interleukin 2

Optimum conditions for the production and assay of guinea pig interleukin-2 (IL-2) have been established. The mitogenic activities of serial dilutions of guinea pig IL-2 preparations were compared in cultures of guinea pig peripheral blood lymphocytes prestimulated for 7 days with phytohemagglutinin (PHA) used at 1 microgram/ml. Parallel log dose-log response curves were used for quantitative comparisons. Optimum IL-2 yields were obtained from cultures of lymph node lymphocytes stimulated for 20 h with Concanavalin A (ConA) at 5 micrograms/ml. Guinea pig T cell lines reactive to mycobacterial antigens were propagated for several months using our IL-2 preparations. The molecular weight of guinea pig IL-2 was estimated to be 30,000 using S-200 gel filtration. The species specificities of guinea pig, human, mouse and rat IL-2s were examined. It was shown that guinea pig T lymphocyte blasts were stimulated only weakly with human IL-2 and not at all with mouse and rat IL-2.     

Interferon induction by the psittacosis agent in Guinea pig leukocyte cultures

High titers of interferon were induced by the psittacosis agent in guinea pig leukocyte cultures. Optimal yields of interferon were produced from freshly prepared guinea pig leukocyte cultures containing 4 × 10⁶ cells per ml inoculated with a psittacosis/cell multiplicity of 1.0 and incubated at 35 C for 24 hr. Leukocytes obtained from 2- to 4-month-old guinea pigs produced 20 times more interferon than leukocytes from 3- to 4-week-old animals. The biological activity of interferon and the kinetics of its production from psittacosis-infected guinea pig leukocyte cultures were similar to those reported for virus-induced leukocyte interferons.     

Met5-enkephalin- and Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers in the pig female reproductive system

The localization of Met5-enkephalin (ME) immunoreactivity in the female genital organs of the rat, guinea pig and pig was studied by indirect immunofluorescence method. In the rat and guinea pig, no ME immunoreactivity was observed in the uterus, fallopian tube or ovary. In the pig uterus and fallopian tube ME-immunoreactive nerve fibers were observed in muscular and submucose layers as well as around the blood vessels. In the pig ovary, ME immunoreactivity was localized in nerve fibers in medullary and cortical parts of the organ. Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity was also studied in the pig uterus, where its distribution was similar to that of ME. The present results suggest that the pig genital organs receive innervation by nerve fibers containing proenkephalin A-derived peptides, which may have a role in modulation of neurotransmission in these organs.     

Similarities and differences between cholinergic systems in the superior colliculus of guinea pig and rat

Summary We studied the distribution of acetylcholinesterase activity and choline acetyltransferase immunoreactivity in the superior colliculus of the guinea pig and the albino rat, using enzyme histochemical and immunohistochemical methods. Choline acetyltransferase-like immunoreactivity was localized in the neuropil throughout the colliculi, but the density of the immunoreactive neuropil varied among layers as well as between species. In the intermediate collicular layers the pattern of choline acetyltransferase immunoreactivity was closely matched by the distribution of acetylcholinesterase activity in guinea pig and rat, confirming our previous findings in the cat. Furthermore, in the guinea pig, but not in the rat, choline acetyltransferase-like immunoreactivity was localized in a prominent population of perikarya of the superficial gray layer.     

Prostaglandin catabolism in the mammalian kidney--histochemical and electrophoretic studies

Using the prostaglandins PGE2, PGF2 alpha and PGB1 as substrates for demonstration of NAD-15-PGDH activity and of 13,14,15-Keto-PGF2 alpha for demonstration of NAD-9-PGDH activity these enzymes were localized in native sections of rat kidney by membrane incubating technique. Both enzymes are active in the same tubular structures of kidney cortex. Simultaneously homogenate supernatant of kidney cortex was separated by micro-electrophoretic technique. NAD-15-PGDH exist in rat kidney in multiple forms, which are varied in number (1 to 3) during postnatal development. One or 2 fractions of NAD-9-PGDH are detected in rat kidney cortex. In the kidney cortex of rabbit and guinea pig also multiple forms of NAD-15-PGDH exist, but not in the kidney cortex of pig. No multiple forms of NAD-9-PGDH are detectable in the kidney of pig, guinea pig, and rabbit. The histochemical and electrophoretical results suggest an important relationship between prostaglandin catabolizing activity and nephrogenesis in the early development of rat kidney. The functional significance of the detected multiple forms of NAD-15-PGDH and the variation in the number of fractions during ontogenesis in rat kidney are yet not clear.     

Metabolism of L-cysteine in guinea pig liver

The metabolism of L-cysteine in guinea pig liver was studied. Guinea pig liver contained 0.45 +/- 0.05 (mean +/- SD) mumol of cysteine, 0.180 +/- 0.080 mumol of 3-mercaptolactate-cysteine disulfide [S-(2-hydroxy-2-carboxyethylthio)cysteine, HCETC], and 8.082 +/- 0.516 mumol of reduced glutathione per g of fresh tissue. The taurine content was 0.912 +/- 0.158 mumol per g of fresh liver. Cysteine dioxygenase (EC 1.13.11.20) activity was several-fold lower than cysteine aminotransferase (EC 2.6.1.3) activity. Lactate dehydrogenase (EC 1.1.1.27) activity was about 10-fold higher than 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) activity. These results indicate that the oxidative metabolism of L-cysteine in the guinea pig liver is not as active as in the rat liver and that L-cysteine, at least in part, is metabolized via the transaminative pathway, in which 3-mercaptopyruvate is partly reduced to 3-mercaptolactate and is utilized to form HCETC.     

The role of novel arachidonic acid metabolites in GnRH action on gonadotrophin release in vitro

Lipoxygenase metabolites of arachidonic acid were shown to stimulategonadotrophin release dose-dependently in rat pituitary cells.The secretory activity of the arachidonate metabolite leukotrieneC₄ (LTC₄ was biphasic and 10-fold more potent than that of thephysiological stimulus gonadotrophin-releasing hormone (GnRH).In pre-labelled, superfused pituitary cells, GnRH dose-dependentlyenhanced the release of [³H]arachidonic acid, which occurredsimultaneously with the secretion of luteinizing hormone (LH).When cells were pre-treated with GnRH for 24 h no response toa further stimulus by GnRH (10^–7 M) could be observedfor either [³]arachidonate nor LH, demonstrating that also indesensitized cells these two mechanisms react similarly. Inaddition, a GnRH antagonist did not affect the release of arachidonateor LH. These results suggest that arachidonic acid may be involvedin the mechanism of GnRH action on gonadotrophins via its lipoxygenasemetabolites and LTC₄ could act as a very potent intracellularstimulus of LH secretion.     

Diadenosine pentaphosphate affects electrical activity in guinea pig atrium via activation of potassium acetylcholine-dependent inward rectifier

Diadenosine pentaphosphate (Ap5A) belongs to the family of diadenosine polyphosphates, endogenously produced compounds that affect vascular tone and cardiac performance when released from platelets. The previous findings indicate that Ap5A shortens action potentials (APs) in rat myocardium via activation of purine P2 receptors. The present study demonstrates alternative mechanism of Ap5A electrophysiological effects found in guinea pig myocardium. Ap5A (10^?4 M) shortens APs in guinea pig working atrial myocardium and slows down pacemaker activity in the sinoatrial node. P1 receptors antagonist DPCPX (10^?7 M) or selective GIRK channels blocker tertiapin (10^?6 M) completely abolished all Ap5A effects, while P2 blocker PPADS (10^?4 M) was ineffective. Patch-clamp experiments revealed potassium inward rectifier current activated by Ap5A in guinea pig atrial myocytes. The current was abolished by DPCPX or tertiapin and therefore was considered as potassium acetylcholine-dependent inward rectifier (I _KACh). Thus, unlike rat, in guinea pig atrium Ap5A produces activation of P1 receptors and subsequent opening of KACh channels leading to negative effects on cardiac electrical activity.     

A smooth muscle active factor isolated from renal cortex of the rabbit.

Homogenates of rabbit renal cortex contained a water-soluble material with striking activity on smooth muscle derived from the rabbit aorta, rat stomach, and guinea pig ileum--but not rat colon or chick rectum. Evidence derived from the spectrum of its pharmacologic activity, the influence of specific competitive antagonists on the smooth muscle responses to the factor, the influence of proteolytic enzymes and its elution position during molecular sieve filtration on Sephadex G-10 made it unlikely that the factor was a prostaglandin, renin, angiotensin, a catecholamine, serotonin, bradykinin, a nucleotide, a small organic product of local metabolism, or a small ion. The agent was not found in extracts of renal medulla, spleen, myocardium, or lung. The smooth muscle response to the factor was blocked by phenoxybenzamine. The renal cortical factor in subthreshold concentration also potentiated responses of the rabbit aorta to angiotensin and norepinephrine. The factor's intrinsic activity and ability to potentiate the smooth muscle actions of endogenous vasoconstrictors make it a candidate as a mediator of smooth muscle responses in a number of states.     

Guinea pig Hageman factor as a vascular permeability enhancement factor.

Hageman factor was purified from guinea pig plasma by successive column chromatography. The guinea pig Hageman factor appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 76,000 daltons by SDS--polyacrylamide gel electrophoresis and 105,000 daltons by gel filtration with a Sephadex G-150 column. Amino acid composition of the guinea pig Hageman factor was similar to that reported for human, bovine, and rabbit Hageman factors. The purified guinea pig Hageman factor, as well as guinea pig plasma, showed strong clotting time correction activity in Hageman-factor--deficient human plasma. The activity could be blocked by the IgG fraction of antiserums against guinea pig Hageman factor raised in rabbits or a goat. The concentration of Hageman factor in guinea pig plasma was determined to be 120 microgram/ml by quantitative radial immunodiffusion assay. The 28,000-dalton active form of Hageman factor (beta-HFa) was prepared from guinea pig Hageman factor by treatment with plasma kallikrein. beta-HFa caused an increase in vascular permeability when injected into guinea pig skin at concentrations as low as 3 x 10(-10) M (0.8 ng). Native, or zymogen Hageman factor did not cause an increase in permeability at concentrations of up to 2 x 10(-7) M. The increased permeability induced by beta-HFa was short lasting, with about a 50% decrease in activity apparent within 6 minutes after intradermal injection. The permeability enhancement activity of beta-HFa was inhibited by pretreatment of beta-HFa with diisopropylfluorophosphate. It may be concluded that active Hageman factor in the interstitial space of guinea pigs acts as a vascular permeability factor of far greater potency than bradykinin.     

Tyrosine hydroxylase-immunoreactivity and its relations with gonadotropin-releasing hormone and neuropeptide Y in the preoptic area of the guinea pig

The present study examines the distribution of tyrosine hydroxylase (TH) immunoreactivity and its morphological relationships with neuropeptide Y (NPY)- and gonadoliberin (GnRH)-immunoreactive (IR) structures in the preoptic area (POA) of the male guinea pig. Tyrosine hydroxylase was expressed in relatively small population of perikarya and they were mostly observed in the periventricular preoptic nucleus and medial preoptic area. The tyrosine hydroxylase-immunoreactive (TH-IR) fibers were dispersed troughout the whole POA. The highest density of these fibers was observed in the median preoptic nucleus, however, in the periventricular preoptic nucleus and medial preoptic area they were only slightly less numerous. In the lateral preoptic area, the density of TH-IR fibers was moderate. Two morphological types of TH-IR fibers were distinguished: smooth and varicose. Double immunofluorescence staining showed that TH and GnRH overlapped in the guinea pig POA but they never coexisted in the same structures. TH-IR fibers often intersected with GnRH-IR structures and many of them touched the GnRH-IR perikarya or dendrites. NPY wchich was abundantly present in the POA only in fibers showed topographical proximity with TH-IR structures. Althoug TH-IR perikarya and fibers were often touched by NPY-IR fibers, colocalization of TH and NPY in the same structures was very rare. There was only a small population of fibers which contained both NPY and TH. In conclusion, the morphological evidence of contacts between TH- and GnRH-IR nerve structures may be the basis of catecholaminergic control of GnRH release in the preoptic area of the male guinea pig. Moreover, TH-IR neurons were conatcted by NPY-IR fibers and TH and NPY colocalized in some fibers, thus NPY may regulate catecholaminergic neurons in the POA.     

Changes of acetylcholinesterase molecular forms in regenerating motor neurons

Axotomy-induced changes of the molecular forms of acetylcholinesterase in the facial nucleus of the rat and guinea pig were investigated. Evidence is presented that facial motoneurons of the guinea pig are capable of synthesizing considerable amounts of 16S acetylcholinesterase, and furthermore that acetylcholinesterase isoenzymes show species differences in their response to axon transection. Three isoenzymes could be separated by velocity sedimentation, which correspond to G1 (4S), G4 (10S) and A12 (16S) acetylcholinesterase. After axotomy, G4 activity was decreased in both species by 40% 2-3 weeks after nerve transection. In the rat, G1 was even further depressed, whereas in guinea pig facial nucleus G1 showed only a slight change. A12 displayed a clear species difference: in the rat, it was decreased to 60% of control 5 days after axotomy. In guinea pig, however, A12 increased dramatically to values of 400-500% of the unoperated control, and maintained elevated levels even 120 days after operation. This result does not agree with the decrease of transmitter metabolism in regenerating nerves and provides support to the hypothesis that acetylcholinesterase in regenerating nerves may have functions different from transmitter hydrolysis.