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Biomedical subjects

R E Garfield

Publications and source records attributed to R E Garfield.

At least 91 records · Page 5Linked to original sources
PubMed1996

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells.

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.

Animals↗
PubMed1995

Gestational changes in oxytocin- and endothelin-1-induced contractility of pregnant rat myometrium.

The mechanical effects of KCl, oxytocin and endothelin-1 on pregnant rat myometrium were examined using intact strips and beta-escin-treated skinned strips. Myometrial tissues from delivering rats were more sensitive to 10.7 mM K+ compared to mid and late gestation. Maximum contractions induced by K+ were obtained at concentrations of 118 mM at mid and late gestation and during delivery. The maximum amplitude of contractions induced by oxytocin and endothelin-1 compared to the 118 mM K(+)-induced contraction increased during the progress of gestation. Maximum contractions induced by oxytocin and endothelin-1 were greater than those induced by 118 mM K+ at delivery, and maximum contractions by oxytocin were larger than those by endothelin-1 during delivery. In 10 microM nifedipine and Ca(2+)-free (containing 2 mM EGTA) solutions, 118 mM K+ contractions were completely abolished; however, both oxytocin and endothelin-1 produced contractions. In Ca(2+)-free solutions, contractions by oxytocin were larger than those by endothelin-1. In skinned myometrial strips, guanosine 5'-O-thiotriphosphate (GTP, 1 microM-1 mM), guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S, 0.1-100 microM) and oxytocin (1 nM-0.1 microM) with 10 microM GTP, but not endothelin-1 with 10 microM GTP increased Ca2+ sensitivity of contractile force.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary.

The present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well-characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20-day-old, 35-day-old, and adult rats, strong Cx43-immunoreactivity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20- and 35-day-old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43-immunoreactivity, whereas Cx43-immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1995

Role of nitric oxide on vasorelaxation in human umbilical artery.

OBJECTIVE: Endothelium-derived relaxing factor (or nitric oxide) is thought to play an important role in control of blood flow in umbilical blood vessels at midgestation compared with term. Previous studies suggest that histamine releases endothelium-derived relaxing factor from umbilical arteries. In this study we intended to clarify the mechanism by which histamine releases endothelium-derived relaxing factor and causes vasorelaxation in human umbilical artery at the midstage (18 to 22 weeks) of gestation. STUDY DESIGN: By means of very thin muscle strips that allow rapid diffusional access of applied drugs (in a few seconds), contractile properties of human umbilical artery were examined. Isometric tensions were measured in response to potassium chloride (39 mmol/L) or caffeine and inhibitory effects of histamine, A23187, glyceryl trinitrate, and 8-bromo-cyclic guanosine monophosphate on these contractions were also examined. RESULTS: Histamine (0.01 to 0.1 mumol/L) did not inhibit 39 mmol/L K(+)-induced contractions of tissues taken at the terminal (38 to 41 weeks) stage of gestation. However, at midgestation histamine (0.01 to 0.1 mumol/L), A23187 (10 mumol/L), and 8-bromo-cyclic guanosine monophosphate (membrane-permeable analog of cyclic guanosine monophosphate, 0.1 mmol/L) inhibited 39 mmol/L K(+)-induced contractions. The inhibitory effects of histamine were antagonized by mepyramine (an H1 antagonist), L-NG-nitro arginine, methylene blue, and Ca++ depletion of the extracellular space but not by cimetidine (an H2 antagonist). Caffeine produced contractions both in the presence and absence of extracellular Ca++ possibly because of the release of Ca++ from intracellular storage sites. Glyceryl trinitrate and 8-bromo-cyclic guanosine monophosphate reduced the caffeine-induced contractions in Ca(++)-free solution. In addition, 10 mumol/L cyclic guanosine monophosphate did not attenuate the Ca++ sensitivity for contractile elements. CONCLUSION: These results suggest that (1) histamine coupled to the histamine H1 receptor increases intracellular Ca++ concentration to stimulate nitric oxide synthase in human umbilical endothelial cells, (2) nitric oxide from endothelial cells activates guanylate cyclase to produce cyclic guanosine monophosphate in the umbilical smooth muscle cells, and (3) cyclic guanosine monophosphate relaxes the umbilical tissues, perhaps as a result of the activation of a Ca++ extrusion system.

Caffeine↗
PubMed1995

Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy.

OBJECTIVES: The aims of the study were to investigate whether a nitric oxide-cyclic guanosine monophosphate relaxation pathway is present in the human uterus and whether it differentially inhibits contractility during pregnancy and labor. STUDY DESIGN: Myometrial strips were obtained from pregnant women who were either in labor or not in labor and from nonpregnant women. Nitrites and cyclic guanosine monophosphate production by the tissues and contractile responses to nitric oxide modifiers were measured. RESULTS: Biochemical assays revealed that nitric oxide (nitrites) and cyclic guanosine monophosphate are generated by the human uterus. Cyclic guanosine monophosphate production by the uterus was increased by L-arginine (the substrate for nitric oxide) and diethylamine/nitric oxide (a nitric oxide donor) and decreased by nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase). Spontaneous contractility in vitro was increased by nitro-L-arginine methyl ester and decreased by diethylamine/nitric oxide, which furthermore produced a dose-dependent inhibition of contractility, and the median effective dose of inhibition in tissues from nonlaboring pregnant patients (1.5 +/- 0.4 mumol/L) is substantially lower than in tissues from laboring pregnant (21.7 +/- 7.4 mumol/L or nonpregnant (20.8 +/- 4.4 mumol/L) women. These studies show that the nitric oxide-cyclic guanosine monophosphate system exists in the human uterus and that it inhibits contractility. Furthermore, the relaxation responsiveness to nitric oxide is elevated during pregnancy and decreased during labor. CONCLUSION: A nitric oxide-cyclic guanosine monophosphate relaxation pathway is present in the human uterus and may be responsible for maintaining uterine quiescence during pregnancy. A decrease in uterine relaxation responsiveness to nitric oxide at term may play a role in the initiation of labor.

Adult↗
PubMed1995

Changes in rat cervical collagen during gestation and after antiprogesterone treatment as measured in vivo with light-induced autofluorescence.

OBJECTIVE: This study was conducted to evaluate use of light-induced autofluorescence for measuring changes in collagen of cervical connective tissue during gestation. STUDY DESIGN: In a conventional laboratory setting light-induced autofluorescence from the cervix of Charles-Dawley timed pregnant rats was measured in vivo. Eight distinct groups at various times of gestation were examined. Measurements were also performed on rats at day 17 of gestation 24 hours after treatment with RU 38486. RESULTS: The amount of light-induced autofluorescence decreases significantly as pregnancy approaches term (means of the arbitrary total counts per spectrum on day 19 vs day 21 = 2.8 x 10(5) vs 1.9 x 10(5), p < 0.01) and reaches its lowest point when the pups are engaged in the cervix (day 19 vs day 22 = 2.8 x 10(5) vs 0.95 x 10(5), p < 0.001) and during delivery (day 19 vs day 22 = 2.8 x 10(5) vs 0.95 x 10(5), p < 0.001) and during delivery (day 19 vs delivery = 2.8 x 10(5) vs 0.83 x 10(5), p < 0.001). Preterm treatment with RU 38486 also caused a significant decrease in the native fluorescence compared with vehicle-injected controls (day 17 control vs day 17 RU 38486 = 2.8 x 10(5) vs 1.5 x 10(5), p < 0.001). CONCLUSION: Significant decreases in cervical collagen at parturition parallel the results of previous studies that used various indirect methods to analyze collagen content. These data support the use of light-induced autofluorescence for detecting and studying the changes in cervical and uterine connective tissue during gestation in vivo.

Analysis of Variance↗
PubMed1995

Relaxant effects of nitric oxide and cyclic GMP on pregnant rat uterine longitudinal smooth muscle.

The purpose of our study was to examine the relaxant effects of sodium nitroprusside (SNP) and cyclic guanosine 3'-5'-monophosphate (cGMP) on pregnant rat myometrium. Using very thin muscle strips, which allows diffusional access of applied drugs (in a few seconds), contractile properties were examined. This technique facilitates study of SNP's effects on uterine contractility as nitric oxide is rapidly inactivated to NO2. SNP did not decrease the amplitudes of 45 mmol/l KCl contractions but decreased spontaneous contractions and 1 mumol/l carbachol contractions. The relaxation of carbachol contractions by SNP were antagonized by methylene blue. In addition, 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP) also inhibited KCl-, carbachol- and oxytocin-induced contractions, however, the relaxant effect of 8-bromo-cGMP was much greater on carbachol and oxytocin contractions than on KCl contractions. Cyclic GMP (1 microM) decreased contractions evoked by various concentrations of Ca2+ and carbachol with 1 mumol/l GTP-gamma S in skinned (membrane-permeable) strips. These results demonstrate that SNP stimulates guanylate cyclase to produce cGMP and that the relaxant effect of cGMP was predominant on pharmaco-mechanical coupling. The cyclic-GMP system may help in maintaining pregnancy and preventing uterine contractions during exposure to stimulating agonists.

Animals↗
PubMed1995

Pre-eclampsia-like conditions produced by nitric oxide inhibition: effects of L-arginine, D-arginine and steroid hormones.

The aim of this study was to establish that inhibiting nitric oxide (NO) production with NG-nitro-L-arginine methyl ester (L-NAME) results in high blood pressure conditions in chronically treated pregnant rats. To validate the model, the effects of L-arginine (the substrate for NO) and D-arginine (the stereoisomer of L-arginine which is not a substrate for NO synthesis) were studied on blood pressure and fetal weights. The effects of a progesterone agonist, promegestone (R5020) and 17 beta-oestradiol were also explored. The NO synthase inhibitor L-NAME was chronically infused s.c. into pregnant rats from day 17 of gestation, either alone or with the simultaneous infusion of L-arginine and injections of sex steroid hormones (promegestone and oestradiol), compounds that may act in the pathogenic pathways of pre-eclampsia. Systolic blood pressure was measured daily. Weight and mortality of pups were recorded immediately after delivery. Blood pressure was elevated significantly in rats treated with L-NAME for only 1 day following infusion; there was a consistent decline during the next 3 days of pregnancy followed by a dramatic and significant rise just prior to delivery and post-partum. Fetal weights were reduced significantly in the L-NAME-treated rats. Co-treatment of L-NAME-infused rats with L-arginine reversed both the increase in blood pressure and the decrease in fetal weights observed with L-NAME alone. R5020, but not oestradiol, also reduced blood pressure and increased fetal weights in the L-NAME-treated animals. NO appears to play essential roles in the regulation of blood pressure during pregnancy, as well as in fetal perfusion and fetal weights at delivery. This study also indicates that progesterone, and not oestrogen, may regulate the vascular adaptations during normal pregnancy. L-Arginine and progesterone agonists like promegestone may have beneficial effects on the high blood pressure levels and reduced fetal weights associated with pre-eclampsia.

Animals↗
PubMed1995

Changes in transcripts encoding calcium channel subunits of rat myometrium during pregnancy.

Extracellular Ca2+ is normally required for myometrial cells to contract. Ca2+ enters muscle cells mainly through voltage-dependent Ca2+ channels (VDCCs) that open in response to action potentials. The synthesis of myometrial VDCCs may change during pregnancy to alter excitation-contraction coupling. We investigated the mRNA levels for the alpha 1- and beta-subunits of the L-type VDCC in rat myometrium to determine whether alterations are associated with term or preterm labor. RNA isolated from myometrial tissues was analyzed by reverse transcription-polymerase chain reaction (PCR) using specific primers designed according to the published sequences of the VDCC subunits. From pregnant rat myometrium, two distinct PCR products were obtained for the alpha 1-subunit: one of the expected size at 372 bp and a smaller at 339 bp. Sequence analysis of the larger product revealed a 99.5 or 88% sequence homology between rat myometrium and rat aorta or rabbit heart, respectively, and the smaller product had an identical sequence to a 33-bp deletion. The two alpha 1-products followed the same trend throughout pregnancy. VDCC alpha 1-mRNA levels increased gradually to 6.9-fold just before labor on day 22 but decreased during labor. However, the beta-subunit mRNA level increased sharply on day 22 and then also declined during labor. Progesterone treatment from day 19 to day 22 inhibited term delivery and prevented the significant increase in alpha 1-mRNA levels. In contrast, antiprogesterone (onapristone, ZK-98.299) treatment on day 17 caused a statistically significant increase in the alpha 1- and beta-VDCC subunit mRNA after 8 and 15 h, respectively, then a decrease during preterm labor at 24 h. We conclude that mRNA levels for the VDCC subunits increase before term and preterm labor but decline during periods when VDCCs are likely at their peaks. The increase in levels of mRNA for VDCC likely reflects changes in expression of VDCCs during periods of term and preterm labor that may facilitate uterine contractility required for this process. Progesterone withdrawal or blockade appears to be responsible for regulating levels of mRNA for VDCC in the myometrium in preparation for labor.

Amino Acid Sequence↗
PubMed1994

Effects of estrogen treatment and inhibition of prostanoid synthesis on myometrial activity and gap junction formation in the oophorectomized ewe.

The objective was to elucidate the functional relationship between estradiol-induced changes in myometrial activity, uterine prostanoid synthesis, and gap junction formation in vivo. The study design was as follows. The effects of inhibition of prostanoid synthesis with naproxen on formation of gap junctions and coordination of electrical and mechanical myometrial activity induced by estradiol-17 beta were investigated in 6 chronically instrumented oophorectomized ewes. Prostanoid metabolites were measured in arterial and uterine venous plasma. Myometrial biopsies were obtained to determine gap junction area. The results were that intraarterial administration of 0.1 mg of estradiol-17 beta was followed by a significant increase in maximum rate of rise of the intrauterine pressure cycles and a significant decrease in conduction time of bursts of electrical activity; this response was not altered by pharmacologic inhibition of uterine prostanoid synthesis using naproxen. Estradiol administration during continuous naproxen infusion increased gap junction area. The conclusion is that improved coordination of myometrial activity and formation of myometrial gap junctions induced by estradiol is not mediated by prostaglandins.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

Some mechanical properties of skinned fibres of pregnant human myometrium.

The properties of contractile elements and intracellular Ca2+ storage sites of pregnant human myometrium were studied by recording the mechanical responses in skinned (saponin-treated and membrane-permeable) fibres. Calmodulin increased the amplitude of contractions induced by Ca2+ and the Ca2+ sensitivity for contractile elements in small myometrium strips, but PGF2 alpha, PGE2, oxytocin, or cyclic AMP failed to produce similar effects. After accumulation of Ca2+ in intracellular Ca2+ storage sites, 10 mumol/l PGF2 alpha, 10 mumol/l PGE2, 30 mmol/l caffeine, and 20 mumol/l InsP3 (inositol-trisphosphate) produced contractions by releasing Ca2+ from storage sites. However, 20 nmol/l oxytocin had no effects under the same conditions. The InsP3 sensitive Ca2+ store was much larger than those of PGs or caffeine. These results suggest that pregnant human myometrium contracts with low Ca2+ by a calmodulin sensitive system. The data also indicate that direct application of PGF2 alpha, or PGE2 into the cells discharges Ca2+ from Ca2+ storage sites and that oxytocin extricates Ca2+ via a pathway involving InsP3 by activation of phosphoinositide turnover. We suggest that these agents induce added contractile responses due to a Ca2+ release mechanism from store sites in addition to the influx of Ca2+ from the extracellular space.

Adult↗
PubMed1994

An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy.

OBJECTIVE: Nitric oxide is synthesized from L-arginine and it causes relaxation of smooth muscle by elevating cyclic guanosine monophosphate levels. We hypothesized that an L-arginine-nitric oxide-cGMP system is present in the uterus and modulates contractility. STUDY DESIGN: Isometric tension of the uterus was measured in vitro from pregnant rats in response to various agents that modulate nitric oxide-cyclic guanosine monophosphate production or action. RESULTS: Major findings are as follows: (1) The substrate and a donor of nitric oxide produced uterine relaxation; (2) inhibitors of the nitric oxide-cyclic guanosine monophosphate pathway blocked the relaxation responses; (3) nitric oxide synthase was localized to several uterine cell types; (4) nitric oxide was produced by the uterus during periods when L-arginine was consumed and citrulline levels increased; (5) effects of nitric oxide substrate on relaxation were mimicked by cyclic guanosine monophosphate; (6) nitric oxide-cyclic guanosine monophosphate responses were decreased during delivery; (7) L-arginine responses were increased by progesterone, and antiprogesterone treatment decreased cyclic guanosine monophosphate-induced relaxations. CONCLUSION: An L-arginine-nitric oxide-cyclic guanosine monophosphate system is present in the uterus and it may regulate relaxation during pregnancy. The inhibitory action of L-arginine and 8-bromo-cyclic guanosine monophosphate was considerably lower during delivery and post partum, indicating that the nitric oxide system may contribute to the maintenance of uterine quiescence during pregnancy, when progesterone levels are elevated, but not during delivery.

Amino Acid Oxidoreductases↗
PubMed1994

Gestational changes in endothelium-dependent vasorelaxation in human umbilical artery.

OBJECTIVE: We intended to clarify whether (1) the amount of endothelium-derived vasorelaxing factor or nitric oxide released by histamine is sufficient to cause relaxation in human umbilical artery at different stages of gestation and (2) the functional role of endothelium-derived relaxing factor changes with the progress of gestation. STUDY DESIGN: By means of a bioassay cascade technique with very thin muscle strips, which allows rapid diffusional access of applied drugs (of the order of a few seconds), contractile properties were examined. RESULTS: At 18 to 22 weeks of gestation histamine produced only a minor contraction, but as gestation progressed the contractile responses increased. L-NG-nitro-arginine greatly enhanced the histamine-induced contractions. Histamine produced a concentration-dependent relaxation during the maintained contraction induced by 39 mmol/L K+ in tissues from 18 to 22 weeks or 30 to 32 weeks but not 38 to 41 weeks of gestation. Thus the concentration-relaxation relationship for histamine showed decreased sensitivity during gestation. The histamine-induced relaxation was enhanced by superoxide dismutase and completely blocked by L-NG-nitro-arginine or mepyramine (H1 antagonist). The concentration-relaxation relationship for the action of glycerol trinitrate relaxation decreased as gestation progressed. In a bioassay cascade an endothelium-intact umbilical artery from 18 to 22 or 38 to 41 weeks of gestation was used as a "donor" and L-NG-nitro-arginine-treated umbilical artery from 18 to 22 and 38 to 41 weeks as a "detector." In the presence of histamine the perfusate that had passed through a donor from 18 to 22 weeks (but not 38 to 41 weeks) attenuated the high K(+)-induced contraction in the detector only from 18 to 22 weeks. CONCLUSION: These results suggest that the amount of endothelium-derived relaxing factor and the sensitivity of smooth muscle to endothelium-derived relaxing factor decreased with the progress of gestation.

Arginine↗
PubMed1994

Uterine contractile responses to endothelin-1 and endothelin receptors are elevated during labor.

The purpose of this study was to determine whether the contractile responsiveness of the rat uterus to endothelin (ET)-1 and myometrial ET-1 receptors is elevated during labor. Uterine contractile responses to ET-1 were measured in isolated uterine strips obtained from rats on Day 18 of gestation, during spontaneous labor at term, and on Days 1 and 2 postpartum. The mean force (millinewtons) generated by cumulative addition of ET-1 (10(-10)-10(-6) M) was measured in vitro. Cumulative dose-response curves were created based upon the contractile activity vs. log doses of ET-1. The values of the estimated pD2, Emax, and slope of the dose-response curves were significantly (p < 0.05) higher in tissues taken during delivery than in tissues from animals on Day 18 of gestation or Days 1 and 2 postpartum. Myometrial ET-1 receptors were also measured during pregnancy and labor. Scatchard analysis of the ET-1 binding to myometrial membranes revealed that receptor levels were significantly (p < 0.05) elevated in delivering animals, without changes in their affinity. Our study shows that the uterus is more sensitive and responsive to the contractile effects of ET-1 during labor, at term, when the receptor density is increased. The change in ET-1 responsiveness at term may play a role in the initiation and/or progression of labor.

Animals↗