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Biomedical subjects

R E Garfield

Publications and source records attributed to R E Garfield.

At least 109 records · Page 6Linked to original sources
PubMed1994

Steroid hormones modulate the production of nitric oxide and cGMP in the rat uterus.

Previously, we demonstrated the presence of an L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the rat uterus and that NO inhibits contractility during pregnancy but not during delivery. In the present study, we investigated the possible role of sex steroid hormones in the regulation of NO synthesis and cGMP generation. NO, measured as nitrite production, and cGMP were determined in full thickness uterine tissues from either pregnant rats on different gestational days or nonpregnant animals after treatment with steroid hormones. NO formation was low in tissues from nonpregnant rats, substantially increased during pregnancy and decreased during labor and immediately postpartum. The cGMP content in the same tissues followed a similar trend. Uterine nitrite production and cGMP levels from animals treated with estradiol and estradiol + progesterone were significantly lower compared to those treated with vehicle or progesterone. These data provide strong evidence that the NO-cGMP system is upregulated during pregnancy to maintain uterine quiescence and a rise in estrogen at term could inhibit this system and thus initiate labor.

Animals↗
PubMed1993

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach.

Heart tissue contains large amounts of the protein encoded by the Ca2+ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms by in situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca2+ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca2+ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.

Animals↗
PubMed1993

Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia.

OBJECTIVES: Preeclampsia is associated with hypertension, fetal growth retardation, and proteinuria. We hypothesized that impaired vascular nitric oxide synthesis during pregnancy may be an important causal factor in preeclampsia. STUDY DESIGN: An inhibitor of nitric oxide synthase, L-nitro-arginine methyl ester, or a nitric oxide donor, nitroglycerin, was infused subcutaneously to rats at a constant rate from day 17 of gestation. Systolic blood pressure, day of spontaneous delivery, weight, and mortality rate of pups were recorded. RESULTS: Systolic blood pressures in rats infused with L-nitro-arginine methyl ester at daily doses of both 25 and 50 mg were significantly elevated compared with controls. This treatment also caused a substantial decrease in the weight of pups, with an increase in mortality rate, without affecting the gestational length. These effects were dose dependent. Nitroglycerin infusion, on the other hand, affected neither the weight and mortality rate of the pups nor the length of gestation. CONCLUSIONS: Infusion of an inhibitor of nitric oxide synthesis during pregnancy causes hypertension and fetal growth retardation, without affecting gestational length. These signs are similar to those of preeclampsia and indicate that an alteration in nitric oxide synthesis may be one of the factors responsible for this disorder. Treatment with nitric oxide inhibitors may be used in an animal model for preeclampsia, to test various therapeutic strategies.

Animals↗
PubMed1993

Gestational changes in L-arginine-induced relaxation of pregnant rat and human myometrial smooth muscle.

OBJECTIVE: We intended to demonstrate the presence of an L-arginine-nitric oxide system in human myometrium and to clarify the mechanisms of action of nitric oxide on rat myometrium during gestation. STUDY DESIGN: By examining very small myometrial muscle strips (approximately 750 muscle cells), characteristic features of contraction of rat longitudinal muscle at the midstage of gestation (day 16) and during delivery at term were determined. RESULTS: Spontaneous contractions were significantly different during delivery compared with the midstage of gestation of rat myometrium. L-Arginine relaxed spontaneous and carbachol-induced, but not potassium chloride-evoked, contractions at both stages. However, much higher concentrations of L-arginine were required during delivery, 8-Bromo-cyclic guanosine monophosphate inhibited spontaneous contractions from concentrations of 1 nmol/L in the midstage of gestation and from 0.1 mmol/L during delivery. In human myometrial tissues L-arginine also inhibited contractions during the late stages of gestation. CONCLUSION: (1) The experimental model is sufficient to compare properties of longitudinal myometrial strips during gestation. (2) In rat and human myometrium an L-arginine-nitric oxide system has an important role in inhibiting uterine contractility and possibly maintaining pregnancy. (3) The relaxing effect of the nitric oxide system is largely because of the voltage-independent action of cyclic guanosine monophosphate systems.

Animals↗
PubMed1993

Connexin43 and gap junction content in the porcine myometrium during the estrous cycle.

The amount of the gap junction protein Connexin43 in myometrium from pig uteri at specific stages of the estrous cycle was studied using 2 different immunochemical methods. Western blot analysis showed the presence of Connexin43 protein in 43 and 47 kD bands at all stages of the cycle. Immunohistochemistry demonstrated only a few small junctional complexes in the myometrium during the luteal phase of the cycle. Gap junctions appeared to increase both in number and size during proestrus and estrus.

Journal Article↗
PubMed1993

Expression of the internal calcium pump in pregnant rat uterus.

Uterine contraction to agents such as oxytocin during labour may utilize Ca2+ sequestered by Ca2+ pump into the sarcoplasmic reticulum (SR). Uterus expresses the SR Ca2+ pump gene SERCA2 as the mRNA splice which encodes the protein SERCA2b. The expression of SERCA2 mRNA and protein was monitored in uteri of day 15 pregnant and delivering rats. The ratio of SERCA2 mRNA to 28S RNA increased by 20% from day 15 to delivery. SERCA2 protein examined by two antibodies increased by 54-55%. Ca2+ dependent acylphosphate intermediate of 115 kD corresponding to SERCA2 also increased 72% during this period. Thus, there is an increase in the SERCA2 expression from day 15 to delivery.

Acylation↗
PubMed1993

Nitric oxide inhibits uterine contractility during pregnancy but not during delivery.

Nitric Oxide mediates various biological phenomena, including vascular smooth muscle relaxation. In the present study, we sought to determine if an L-arginine nitric oxide-relaxation system is present in the uterus and if it modulates contractility during pregnancy. The substrate and a donor of nitric oxide and nitric oxide gas caused substantial relaxation of the spontaneous contractility of tissues from the rat uterus in vitro during pregnancy. Inhibitors of nitric oxide synthase and soluble guanylate cyclase reversed the relaxation effects of L-arginine. Nitric oxide was produced by the uterus in organ culture. Relaxation effects of L-arginine on the pregnant rat uterus were diminished at the time of spontaneous labor and postpartum. Nitric oxide production was also substantially reduced during labor. These results show that an L-arginine-nitric oxide-relaxation system is present in the uterus and it inhibits contractility during pregnancy but not during labor.

Animals↗
PubMed1992

Polymerase chain reaction assay of mRNA using 28S rRNA as internal standard.

A method is developed for PCR assay of mRNA using 28S rRNA as internal standard and its applicability is reported for 4 types of mRNA. It consists of reverse transcription of total RNA using primers selective for 28S rRNA and the message of interest. Routinely the 28S reaction mix is diluted 20,000-fold and the dilution error is minimized by adding 10 microCi of 3H2O before the dilution and determining the level of 3H before and after the dilution. Aliquots from the diluted 28S mix and the undiluted reverse transcripts are co-amplified by PCR in the presence of 32P-dATP. The samples are analyzed by electrophoresis, bands detected by autoradiography and in each lane the ratio is determined between cpm in the band of interest and cpm in the 28S band. The ratio is a measure of the level of mRNA in the sample and can be used to monitor changes in mRNA during treatments, development and pathogenesis.

Animals↗
PubMed1992

Premature labour induced by cortisol in the unrestrained pregnant rabbit.

The relationship between electrical activity (EMG) of the uterus, progesterone levels and gap junctions was studied in pregnant rabbits delivering either prematurely (cortisol-induced) or spontaneously at term. Electrodes were implanted in the myometrium and a catheter was inserted into the jugular vein for continuous infusion of saline or cortisol from gestational age 25 days. A tether and swivel system was used to connect the electrodes and catheter to the recording unit thus allowing protection of the wires and catheters while enabling the rabbit to move freely. The EMG was recorded continuously from day 25 of pregnancy until animals either delivered or were sacrificed at predetermined days of gestation. Both rabbit groups showed a similarity in patterns of EMG, progesterone levels and gap junction formation. We conclude that preterm labour induced by cortisol mimics normal term labour.

Analysis of Variance↗
PubMed1992

Modulation of cell-to-cell coupling between myometrial cells of the human uterus during pregnancy.

OBJECTIVE: The purpose of this study was to investigate changes in cell-to-cell coupling of human myometrium during pregnancy to assess the presence and permeability of gap junctions. STUDY DESIGN: To evaluate the coupling, input resistance was measured and intercellular spread of Lucifer yellow was observed with microelectrode techniques in intact myometrial preparations from four nonpregnant women, 13 women not in labor, and three women in labor. Octanol, isoproterenol, and dibutyryl adenosine 3',5'-cyclic monophosphate were applied to the preparations to assess their effects on cell-to-cell coupling. RESULTS: Input resistance of myometrial cells was decreased (p less than 0.001) and intercellular spread of Lucifer yellow was increased during pregnancy. Octanol, isoproterenol, and dibutyryl adenosine 3',5'-cyclic monophosphate rapidly and reversibly increased input resistance (p less than 0.001 for all these agents) and blocked Lucifer yellow spread in tissues from pregnant patients. CONCLUSIONS: Cell-to-cell coupling between human myometrial cells is spontaneously improved during pregnancy because of the presence of gap junctions. The coupling is rapidly and reversibly decreased by octanol, isoproterenol, and dibutyryl adenosine 3',5'-cyclic monophosphate as a result of decreased permeability of gap junctions. These two methods of modulation of gap junctions are suggested to be major mechanisms for control of myometrial contractile activity in the human uterus during pregnancy.

1-Octanol↗
PubMed1992

Studies of connexin 43 and cell-to-cell coupling in cultured human uterine smooth muscle.

OBJECTIVE: The aim of this study was to assess the presence and the permeability of gap junctions between human uterine smooth-muscle cells in culture. STUDY DESIGN: The uterine smooth muscles obtained from term-pregnant women were cultured. The presence of gap junction was evaluated by immunocytochemistry with gap junction protein antibodies and by measuring input resistance and intercellular spread of lucifer yellow. These measures also evaluated the permeability of gap junctions. Octanol, isoproterenol, dibutyryl cyclic adenosine monophosphate and forskolin were applied to the cultures to assess their effects on the permeability of gap junctions. RESULTS: During culture, immunocytochemical staining of gap junction protein (connexin 43) was increased and input resistance was decreased on day 2 of culture versus day 21 (18.4 +/- 7.87 M omega day 2; 3.8 +/- 1.76 M omega, day 21; p < 0.001). However, the decrease in input resistance was related to cell density rather than time in culture (16.4 +/- 5.01 M omega, single cells on days 1 and 2; 5.3 +/- 2.35 M omega, high-density cultures on days 1 and 2; p < 0.001). Octanol increased input resistance and intercellular spread of lucifer yellow in confluent cultures; isoproterenol, dibutyryl cyclic adenosine monophosphate, and forskolin did not. CONCLUSIONS: The increased staining of connexin 43 and the decreased input resistance during culture are evidence of elevated number of gap junctions between cells. The rapid and reversible increase in input resistance and decrease in spread of lucifer yellow by octanol are the result of decreased permeability of gap junctions. These two methods of modulation of gap junctions in human uterine smooth muscles are thought to be major mechanisms for the control of uterine contractility.

Cell Communication↗
PubMed1992

Effects of antiprogesterones on myometrial cell-to-cell coupling in pregnant guinea pigs.

We used intracellular microelectrodes to investigate the effects of the antiprogesterone (AP) compounds RU 486 and ZK 299 on cell-to-cell coupling in the guinea pig myometrium during pregnancy. The input resistance (Ro) of myometrial cells was high in nonpregnant tissues (44.6 +/- 6.39 M omega), but decreased by midgestation (Day 44 or 45 of gestation; 22.9 +/- 3.17 M omega), and was lowest at term (between 17.7 +/- 2.90 m omega and 13.1 +/- 4.34 M omega on Days 59-69). Treatment with the AP RU 486 or ZK 299 in three groups of midgestational animals reduced Ro to a similar level within 24 h. Lucifer Yellow (LY) was injected into smooth muscle cells as a direct but qualitative measure of metabolic coupling. In term and AP-treated animals, LY spread rapidly to neighboring cells within 60 sec, but little spread occurred in midgestational control tissues and no spread was seen even after 10 min in nonpregnant tissues. This correlation of decreased Ro (implying increased electrical coupling) with the development of extensive spread of LY indicates increased electrical and metabolical coupling between myometrial cells during labor. These data show that myometrial smooth muscle cells of guinea pigs are moderately well coupled before the onset of labor, and the coupling increases further, just prior to spontaneous delivery or due to treatment with APs. These events may be required for synchronizing and coordinating the electrical, metabolic, and contractile activity of labor.

Animals↗
PubMed1992

Intracellular cyclic AMP concentration modulates gap junction permeability in parturient rat myometrium.

We investigated whether cell-to-cell coupling between myometrial cells of parturient rats is influenced by intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration. To evaluate the coupling, we measured input resistance (Ro) and injected Lucifer Yellow (LY) using microelectrode techniques. The intercellular spread of the dye was then observed. Longitudinal muscle strips from rat myometrium were exposed to isoproterenol, forskolin, or dibutyryl cAMP (DB-cAMP) to elevate cAMP. Isoproterenol (10(-11)-10(-6) M) and DB-cAMP (10(-5)-10(-3) M) hyperpolarized the resting membrane potential (Em) and increased Ro in a dose-dependent fashion. Forskolin (10(-6) M) also hyperpolarized Em and increased Ro. When LY was injected into a single cell, LY spread rapidly and extensively to neighboring cells in parturient control tissues, while LY transfer was completely blocked by any of the three agents at high concentrations. The increased Ro and blocked transfer of LY owing to these agents indicate that the cell-to-cell coupling was decreased both electrically and metabolically. Myometrial cells of parturient rats show increased number and size of gap junctions (GJs). The rapid and reversible decrease in coupling is interpreted to reflect the reduced permeability of GJs between the muscle cells because of an elevation of cAMP. Control of GJ permeability by this second messenger may be important for the physiological regulation of intercellular coupling and the extent of synchronizing and coordinating electrical, metabolic, and contractile activity in the uterine wall during pregnancy and parturition.

Animals↗
PubMed1992

Are gap junctions necessary for cell-to-cell coupling of smooth muscle?: An update.

Earlier, it was questioned whether gap junctions (GJs) were necessary for cell-cell communication in smooth muscle, and GJs were not seen in some smooth muscles. We reexamined this question in the myometrium and in intestinal smooth muscle, in light of current knowledge of the presence and function of GJs. In the uterus, numerous studies show that an increase in GJ number is associated with the onset of delivery and is required for effective parturition. In all cases, this increase in GJ number and the changes in uterine contractility were correlated with increased electrical and metabolic coupling. Evidence for the much smaller, but detectable, degree of electrical coupling in the preterm uterus is explained by the small (but again detectable) number of GJs present. In the intestine, GJs are readily detected in the circular muscle layer but have not been described in the adjacent longitudinal layer. While our immunohistochemical studies failed to detect GJs in the longitudinal layer, this may not be adequate to prove their absence. Therefore, current knowledge of GJ number and function is adequate to explain cell-cell coupling in the uterus. Although it remains uncertain whether GJs are absent from the longitudinal muscle of the intestine, there is no definitive evidence that cell-cell coupling can occur by means other than GJs.

Animals↗
PubMed1992

Ca pump messenger RNA expression in pregnant rat uterus.

Rat uterus expresses transcripts for the plasma membrane Ca pump PMCA1 and for the sarcoplasmic reticulum Ca pump SERCA2. Uterus contains two mRNA isoforms for PMCA1, one encoding a cyclic nucleotide insensitive Ca pump (PMCA1a) and the other encoding a cyclic nucleotide sensitive pump (PMCA1b). The relative ratio of mRNA to 28S RNA for PMCA1a or PMCA1b and the amount of poly A+ RNA per microgram total RNA obtained from uteri of rats pregnant for 15 days and for parturient rats was similar but the ratio of SERCA2 mRNA to 28S RNA was significantly higher in the parturient rats.

Animals↗