Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

Summary In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.     

Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

Phospholipase A(2) (PLA(2)) is activated in spermatozoa in response to progesterone and Ca(2+) ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA(2). We investigated whether PLA(2) is involved in ZP-stimulated acrosomal exocytosis, if Ca(2+) is required for activation of PLA(2), and signal transduction pathways modulating PLA(2) using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca(2+) medium with [(14)C]choline chloride or [(14)C]arachidonic acid and were then exposed to millimolar Ca(2+) and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca(2+) and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA(2) activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA(2) inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca(2+) or in medium with millimolar Ca(2+) and EGTA or La(3+) resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a G(i) protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA(2) plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA(2) activation requires Ca(2+) internalization, and that PLA(2) activation is regulated by signal transduction pathways involving G proteins and DAG.     

Pulmonary responses to phospholipase A2 in the perfused guinea pig lung

We examined the effect of phospholipase A2 (PLA2; Naja naja) challenge on pulmonary hemodynamics, airway constriction, and fluid filtration in isolated Ringer-perfused guinea pig lungs. Intratracheal PLA2 (10-100 U) produced dose-dependent increases in pulmonary arterial pressure, intratracheal pressure, and lung weight, although intravenous PLA2 administration had no effect on monitored variables. Morphological features indicative of airway constriction and pulmonary edema were observed by light microscopy. PLA2-induced increases in intratracheal pressure and/or lung weight were attenuated to varying degrees by pretreatment with indomethacin (1 microM, a cyclooxygenase inhibitor), ICI-198,615 (1 microM, a leukotriene D4 receptor antagonist), and WEB 2086 (1 microM, a platelet-activating factor antagonist). PLA2-induced increases in pulmonary arterial pressure and intratracheal pressure were also reduced in lungs removed from animals pretreated with dexamethasone (50 mg/kg ip for 2 days; a steroidal antiinflammatory agent). Pyrilamine (1 microM, a histamine1-receptor antagonist) and Takeda AA861 (1 microM, a delta 5-lipoxygenase inhibitor) did not produce significant inhibitory effects on PLA2-induced pathophysiological changes. Intratracheal instillation of high-dose platelet-activating factor (50 micrograms) or lysophosphatidylcholine (100 micrograms) produced gradual increases in intratracheal pressure and lung weight, but these changes were not as large as those induced by PLA2. Thus these studies suggest that resident cell populations associated with airways may play an important role in PLA2-induced pathophysiological changes in the perfused guinea pig lung. These PLA2-induced effects are most likely partially mediated by generation of eicosanoids and platelet-activating factor.     

Oxytocin receptors in guinea pig myometrium near term and during labor

Oxytocin receptors in myometrium of women, rats, and rabbits rise markedly before the onset of labor, suggesting a role in the initiation of labor. In guinea pigs, a previous study reported no such rise by one-point determination of oxytocin binding. The purpose of this study was to use a more rigorous method to determine whether the binding characteristics of myometrial oxytocin receptors change in relation to labor in guinea pigs. Competitive binding studies were carried out in microsomes from inner and outer myometrium between 42 days of gestation and labor. Binding to analogs was also tested. Data were analyzed with affinity spectra and LIGAND. Oxytocin bound to one site with a dissociation constant of 6.3 +/- 0.65 x 10(-9) M. Binding capacity was 1.0 +/- 0.1 x 10(-12) mol/mg protein. The Hill coefficient was near unity. No significant changes occurred with gestation or labor in dissociation constant, binding capacity, or Hill coefficient (all P >/= 0.2, nested ANOVA). Binding capacity was higher in the outer than in the inner layer (1.2 +/- 0.2 vs. 0.8 +/- 0.1 x 10(-12) mol/mg protein, P = 0.02), but the dissociation constants were similar. Differences existed in the dissociation constants of the analogs tested. The main conclusion is that oxytocin receptors are unlikely to have a regulatory role in the initiation of labor in guinea pigs.     

Changes in phosphatidylinositol phospholipase C isoenzymes and in their association with GTP gamma S-binding activity in guinea pig uterine smooth muscle during pregnancy.

The nature distribution and associated GTP gamma S binding activity of phosphatidylinositol phospholipase C (PI-PLC) has been studied in non-pregnant and pregnant guinea pig uterine smooth muscle. Cytosolic fractions partially purified by Q-Sepharose and heparin-Agarose chromatography show two isoenzyme forms, one with an apparent molecular weight of 58 kD that crossreacts with PI-PLC alpha and a has Km for phosphatidylinositol of 292 +/- 72.6 microM, designated alpha, and a form that has an apparent molecular weight of 86 kD and a substrate Km of 54 +/- 20 microM designated delta. Approximately 80% of the total PI-PLC activity was recovered in the cytosolic fraction and this increased 8-10 fold for both isoenzymes from the non-pregnant to the late pregnant uterus and the proportion of the alpha isoenzyme increased from approximately 40% to 55% of the total. PI-PLC alpha but not delta activity had GTP gamma S binding activity associated with it after Q-Sepharose or heparin-Agarose chromatography. This associated activity accounted for 2% of the total GTP gamma S-binding activity in the non-pregnant uterus and 31% of that in the near-term uterus. On separation of the PI-PLCa-GTP gamma S-binding complex by gel filtration on Sephacryl S200 gave two peaks one of 118 kD accounting for two-thirds of all the binding and two-thirds of the enzyme activity and a 58 kD peak. The 118 kD peak could not be separated by treatment with 0.5% cholate, but in this form enzyme activity was protected from detergent inactivation found with the 58 kD form. In sodium dodecyl sulphate polyacrylamide-gel electrophoresis PI-PLC alpha was released from the 118 kD complex and showed an apparent molecular weight of 61.5 kD. All the activity in the residual membrane fraction could be released by washing with buffer followed by, 2 M KCl and then 2 M KCl plus 0.5% cholate. This released isoenzyme forms that appeared identical to those in the cytosolic fraction and with GTP gamma S-binding activity associated with PI-PLC alpha. It is concluded that in the near term guinea pig uterus there is a dramatic increase in the capacity for inositol polyphosphate production. Moreover the dramatic increase in GTP gamma S-binding activity associated with PI-PLC alpha implies large changes in the extent and possibly nature of the putative G-protein activation of this pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Flavonoids differentially inhibit guinea pig epidermal cytosolic phospholipase A2

Cytosolic phospholipase A2 (cPLA2) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA2 may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA2 in guinea pig epidermis. The epidermal cPLA2 is Ca2+-dependent, active at micromolar concentration of Ca2+ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA2, while 12-epi-scalardial (a sPLA2 inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA2, while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA2 activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA2 activity.     

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.     

Placental fatty acids in the guinea pig during pregnancy

Prostaglandins are involved in different stages of reproduction. In respect to the placental metabolism of prostaglandins in the Guinea pig, we have studied the composition of Guinea pig placenta in free and total fatty acids. The arachidonic and dihomo-gamma-linolenic acids, precursors of prostaglandins of series 2 and 1, and the linoleic acid were quantified at different gestational ages using a gas-chromatography capillary column technique. Only the linoleic acid shows a significant increase at the end of gestation.     

Changes in the LHRH-immunoreactivity of hypothalamic structures during late pregnancy and after parturition in the guinea pig

Summary LHRH-immunohistochemistry and radioimmunoassay of the gonadotropins were used to demonstrate changes in the LHRH content of the hypothalamus during late pregnancy and after parturition in the guinea pig. Immediately after parturition a decrease in the amount of LHRH-associated fluorescence was observed in the medial preoptic area. Only 12 h later similar changes were found in the median eminence. In the precommissural region no obvious changes were noted in the quantity of LHRH-immunoreactive terminals and synapses. The radioimmunoassay of the gonadotropins shows an increase of LH shortly after parturition which reaches the level of a preovulatory rise, whereas the amount of FSH does not vary significantly. The role of the medial preoptic area and the OVLT in the regulation of the estrous cycle through LHRH is discussed.This work was performed under a postdoctoral fellowship at the INSERM unité 156, Lab. d'Histologie, Faculté de Médecine, Lille, France     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Contractile activity of Trimeresurus flavoviridis phospholipase A2 on guinea pig ileum and artery.

Trimeresurus flavoviridis phospholipase A2 (PLA2) induced strong contractions of the smooth muscles of guinea pig ileum and artery in a concentration-dependent manner (10(-10)-10(-6) M). When the same dose of PLA2 was administered in repetition to the ileal preparation, the contraction diminished progressively and was no longer recovered even by consecutive washings. The enzymatically inactive derivative of PLA2, in which His-47 was p-bromophenacylated, was unable to elicit contraction. Also, no activity was observed when the Ca(2+)-free medium was used. The contraction induced by PLA2 was inhibited completely by 1.0 x 10(-6) M indomethacin, but not by nordihydroguaiaretic acid. These results imply that the PLA2-induced contraction is due essentially to the hydrolytic action of the enzyme against phospholipid membranes to liberate arachidonic acid that is then converted to pharmacologically active prostaglandins. In guinea pig artery, PLA2 caused both contraction and relaxation.     

Steroid-induced proteins secreted by the uterus of the guinea pig.

This study was conducted to identify proteins synthesized and secreted de novo by the guinea pig uterus. Uterine samples were obtained from cycling, late-pregnant as well as ovariectomized and steroid-treated guinea pigs and cultured with either L-[3H]leucine or L-[35S]methionine. Two-dimensional SDS-PAGE of culture medium followed by fluorography was used to determine proteins synthesized and secreted de novo during a 24-h incubation period. Two complexes of estradiol-stimulated proteins (ESP) were detected. Each complex was composed of 5-7 unique proteins with slightly different isoelectric points. The higher molecular-weight complex had a molecular weight of 65,000-60,000 and an isoelectric point range of 5.2-6.1. The lower molecular-weight complex had a molecular weight of 60,000-55,000 and a similar range of isoelectric points. The two complexes of ESP were not observed in medium of explants from animals that received placebos, were late-pregnant, or were treated with progesterone only. Progesterone administered in combination with estradiol enhanced production of both complexes of ESP to similar degrees. Neither complex of ESP was secreted by the explant culture in the presence of tunicamycin, suggesting that the proteins are glycosylated. These findings demonstrate that the uterus of the guinea pig produces two unique complexes of proteins in response to estradiol stimulation, and all results are consistent with the hypothesis that ESP are contained in the carbohydrate-rich secretory granules of endometrial gland cells.     

Purification and characterization of a soluble phospholipase A2 from guinea pig lung

Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.     

Multiple biopsies of guinea pig mammary glands during pregnancy and lactation.

Mammary gland biopsies were obtained from 39 guinea pigs during late pregnancy, lactation and weaning. Up to eight samples, each weighing 60--200 mg, were taken from each animal in two independent studies. No mortalities resulted, and no interference with lactation or suckling was observed.     

Electrical coupling in rat myometrium during pregnancy.

Gap junctions were found in rat myometrium only during parturition of abortion. A comparison was made between the values of the length constants obtained from longitudinally cut strips of rat myometrium at the midterm stage of pregnancy and during parturition. No significant difference was found between these values. No significant difference was found between length constants measured in myometrium from rats near term (22 days) and those during parturition, or between midterm myometrium and myometrium taken from animals aborting at midterm following ovariectomy. It was concluded that the appearance of gap junctions in parturient myometrium does not alter the spread of passive current in the longitudinal axis of the cells in these tissues. It is suggested that gap junctions are not required for spread of passive current, and that other structures may provide an alternate path.     

Inhibition of phospholipase A2 activity in guinea pig eosinophils by human recombinant IL-1 beta.

The effect of human rIL-1 beta on the release of arachidonic acid (AA) and on the phospholipase A2 (PLA2) activity in guinea pig eosinophils was investigated. Stimulation of [3H]AA-labeled eosinophils with the ionophore A23187 resulted in a time and concentration-dependent release of AA in parallel to hydrolysis of endogenous phosphatidylcholine (PC). Both events were abrogated by the chelation of intracellular free calcium, but not by its depletion from the medium, suggesting that the ionophore-induced AA release involves a PLA2 activity dependent on the mobilization of intracellular calcium. Addition of human rIL-1 beta (0.01 to 100 ng/ml) to eosinophils for 15 min had no effect on the release of AA induced by the ionophore. However, prolonged incubation with human rIL-1 beta (30 to 180 min) inhibited in a concentration- and time-dependent manner the release of AA and the hydrolysis of phosphatidylcholine in ionophore-stimulated eosinophils. Our results also showed that eosinophil homogenates contain a calcium-dependent PLA2 whose activity was markedly reduced when eosinophils were pretreated with human rIL-1 beta. The inhibition was time and concentration dependent and was observed in the presence of calcium and phospholipid excess. Finally, studies with Fura-2-loaded eosinophils showed that the ionophore A23187 stimulated an increase in intracellular calcium concentration that was not altered by pretreating the eosinophils with human rIL-1 beta. These results suggest that human rIL-1 beta inhibits the release of AA by eosinophils via the inhibition of a PLA2 activity and through a calcium-independent mechanism. Inhibition by human rIL-1 beta required a prolonged incubation (30 to 180 min) and was observed after its removal from the medium, suggesting that human rIL-1 beta did not interact directly with the PLA2 itself, but with a metabolic process involved with the regulation of its activity in eosinophils.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Changes in permeability of fetal guinea pig skin during gestation

Permeability of fetal skin to tritiated water was measured in vitro using samples taken from the back and flanks of 21 guinea pig fetuses whose gestational age ranged from 30 to 67 days (term = 68 days). From 30 to 45 days, fetal skin was relatively permeable to water, with a permeability coefficient for unidirectional, diffusional transfer of labelled water that averaged 0.372 +/- 0.041 (SEM) X 10(-4) cm/s. Then during a 5-10 day interval, the measured permeability coefficient decreased abruptly to very low and barely detectable levels. These changes took place at the time during gestation when others have shown the skin becomes keratinized and growth of new hair follicles is completed. Thus these findings are consistent with a relatively free exchange of water between amniotic fluid and fetal interstitium across the skin during the first two-thirds of gestation and then with further maturation an abrupt functional separation between these fluid compartments during the last third of gestation.