Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes, as revealed by proteomic analysis.

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.

The association between meconium and the production and reabsorption of lung liquid and lactate loss by in vitro lungs from fetal guinea pigs.

OBJECTIVE: We sought to use an in vitro approach to determine relationships between meconium and fetal lung liquid production and to relate meconium to possible problems in lung liquid removal at birth. STUDY DESIGN: Near-term fetal guinea pigs were divided according to the level of spontaneously occurring meconium (no meconium, light meconium, or heavy meconium). Their lungs were maintained in vitro in Krebs-Henseleit saline solution for 3 hours. Lung liquid production or reabsorption was measured by a dye-dilution method, and loss of lactate to the lung liquid and outer Krebs-Henseleit solution was monitored. Reabsorptions were investigated by activating powerful responses with dinitrophenol during the middle hour. RESULTS: During the first hour, lungs from meconium-free fetuses produced liquid at 1.25 +/- 0.12 mL/kg(-1) body weight. h(-1); rates from light or heavy meconium groups were not significantly different (n = 18). Similarly, total lactate loss was not significantly different between the meconium-free and light-meconium groups but was twice as high in heavy-meconium preparations (73.68 +/- 10.60 micromol/L. g(-1) dry lung tissue. h(-1); P <.025, analysis of variance). The meconium-free and heavy-meconium groups continued to produce fluid, with no significant change throughout the 3 hours of incubation; lactate losses fell slightly. Therefore there were no problems with fluid production with meconium present, but the high-lactate losses with heavy meconium suggested long-term intrauterine hypoxia. Dinitrophenol produced powerful reabsorptions in lungs from meconium-free fetuses (-0.85 +/- 0.35 mL. kg(-1) body weight. h(-1); P <.005, analysis of variance; P <.0005, regression analysis) but failed to do so in heavy-meconium fetuses (n = 36). Lactate losses rose 2-fold in both groups (P <.005 to P < 0.0005, analysis of variance and regression analysis), despite already elevated losses with heavy meconium (n = 12). Therefore, in heavy-meconium fetuses, dinitrophenol affected metabolic pathways but did not activate fluid reabsorption, suggesting damage to reabsorptive mechanisms. CONCLUSION: Unless major airways are blocked, meconium is not associated with reduced fetal lung liquid production, which can cause poor lung development, but there may well be poor fluid removal after birth because of compromised reabsorptive mechanisms, which are unlikely to be helped by possible hormonal intervention.

Fluid production by in vitro lungs from near-term fetal guinea pigs: effects of cortisol and aldosterone.

Lungs from near-term fetal guinea pigs (62 +/- 1 days of gestation) were supported in vitro for 3 h and fluid production was determined by a dye dilution method (Blue Dextran 2000). Three groups of control preparations (N = 6 for each group) showed no changes during incubation. However, cortisol or aldosterone placed in the outer saline during the middle hour caused profound reductions in fluid production. Cortisol at 10(-6) or 10(-8) mol/l reduced production 80.3 +/- 10.8% and 47.8 +/- 20.5%, respectively (p < 0.05-0.001; N = 6 for each group); at 10(-10) mol/l it failed to affect production significantly. Aldosterone was effective at lower concentrations (N = 12). At 10(-11) mol/l it reduced production 67.1 +/- 10.0% (p < 0.01-0.001); at 7 x 10(-10) mol/l it produced similar effects. In contrast, there were no significant changes after treatment with 10(-11) mol/l aldosterone together with an aldosterone antagonist (5 x 10(-8) mol/l spironolactone; N = 6). Spironolactone alone was without effect (N = 6). The highest steroid concentrations tested corresponded to plasma concentrations in the guinea pig at delivery; therefore, it is suggested that both steroids may have a role in reducing lung fluid production close to birth in this species.

Lung liquid production by in vitro lungs from fetal guinea pigs: effects of arginine vasopressin and arginine vasotocin.

Lungs from near-term fetal guinea pigs were supported in vitro for 3 h; lung liquid production rates were measured by a dye dilution technique. Seventy preparations were used to study the effects of arginine vasopressin (AVP) placed in the outer saline for the middle hour, at concentrations reported at birth [fetuses 61 +/- 2 days of gestation; 94.7 +/- 16.2 g (SD) body weight]. At 1200 microU/ml, AVP arrested fluid production (rates, successive hours, 3.03 +/- 0.60, 0.50 +/- 0.14 and 0.02 +/- 0.08 ml/kg body weight per h; falls significant, P < 0.01-0.0005). At 600, 300 and 100 microU/ml there were significant but smaller reductions. Reabsorptions were seen in 8 preparations given 600-1200 microU/ml, AVP. Preparations given 10 microU/ml AVP, AVP carrier or control saline showed no significant change. The responses (% reductions during treatment), were linearly related to the log concentration of AVP (r = 0.99); theoretical threshold, 8 microU/ml). Increasing treatment to 2h did not increase final responses. Preparations from 5 fetuses > 120 g body weight showed significantly greater responses (P < 0.025) [fetuses 64 +/- 2 days of gestation; 135.1 +/- 18.6 g (SD) body weight]. 10(-6) M amiloride abolished responses to AVP [fetuses 62 +/- 1 days of gestation; 93.4 +/- 18.5 g (SD) body weight, n = 30; rates, succeeding hours; AVP alone, 1.78 +/- 0.22, 0.48 +/- 0.09, 0.16 +/- 0.99 (P < 0.01-0.0005); AVP with amiloride, 1.15 +/- 0.07, 0.93 +/- 0.10, 0.86 +/- 0.08 (no significant fall) ml/kg body weight per h]. Thirty-six preparations treated with arginine vasotocin (AVT, 10-600 microU/ml) showed closely similar responses to those from AVP. These studies extend results to fetal guinea pigs, and show that AVP, at concentrations reported at delivery, can slow lung liquid production or cause reabsorption by a direct action on the lung. The effect increases close to term, and is due to activation of amiloride-sensitive Na+ channels.

Reabsorption of lung liquid produced by 2,4-dinitrophenol in in vitro lungs from fetal guinea pigs.

Lungs from near-term fetal guinea pigs (62 +/- 2 days of gestation) were supported in vitro for 3 h, and lung liquid production was measured by a dye dilution technique. Twelve untreated preparations produced fluid at 1.54 +/- 0.29 mL.kg-1 body weight.h-1 during the 1st h, with no significant changes in later hours. Twelve preparations treated with 2 x 10(-4) M 2,4-dinitrophenol (DNP) showed strong reabsorptions (significant, ANOVA and regression analysis); total loss of lactate from the preparations doubled (significant, same tests). In 12 additional preparations, increasing DNP fivefold did not abolish reabsorption; results resembled those at the lower concentration. Amiloride at 10(-6) M abolished reabsorptions after 2 x 10(-4) M DNP, although fluid production still halted (n = 6; reductions significant, same tests). Amiloride alone had no effect (n = 6); untreated controls showed no change (n = 6). Similarly, 10(-4) M sodium iodoacetate virtually abolished reabsorptions after 2 x 10(-4) M DNP, although fluid production still stopped (n = 6; reductions significant, same tests). Iodoacetate alone only reduced fluid production (n = 6; significant, same tests); untreated controls showed no change (n = 6). The results suggest that reabsorptions seen after inhibition of oxidative processes depend on amiloride-sensitive Na+ channels and glycolytic metabolism.

Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production.

Somatostatin-like immunoreactivity was measured by radioimmunoassay with a monoclonal antibody in lungs from perinatal guinea pigs (62 +/- 2 days of gestation). Fetuses delivered by Caesarean section and dissected before breathing showed 4748 +/- 758 pg/lung (n = 25). Fetuses allowed to breathe (neonates) showed marked increases in activity: 7629 +/- 1355 pg/lung (n = 12) after breathing 30 seconds, and 10729 +/- 1064 pg/lung (n = 6) after breathing 3 minutes (2.3-fold increase, P < 0.005). Values then declined (5203 +/- 1050 pg/lung (n = 9) at 30 minutes; 1458 +/- 105 pg/lung (n = 4) at 60 minutes). Changes were similar in pg/g wet tissue. HPLC characterized the immunoreactive peptides as somatostatin-14 (SS-14) and somatostatin-28 (SS-28) in both fetuses and neonates (n = 11). SS-28 made up only 13.7 +/- 1.7% of the activity; this percentage did not change with breathing. The effects of synthetic SS-14 on lung liquid production were investigated in in vitro lungs from 42 fetal guinea pigs. All 21 preparations immersed in 10(-5)-10(-7) M SS-14 during the middle hour of 3 h incubations reduced production, often approaching zero after treatment (rates, ml/kg body weight per h, succeeding hours: 10(-5) M (n = 9), 3.09 +/- 0.68, 0.93 +/- 0.39, -0.05 +/- 0.60 (fall significant during and after treatment, P < 0.025-0.005); 10(-6) M (n = 6), 3.06 +/- 0.68, 1.29 +/- 0.58, 0.36 +/- 0.38 (P < 0.05-0.005); 10(-7) M (n = 6), 1.96 +/- 0.66, 1.11 +/- 0.34, 0.64 +/- 0.28 (P < 0.05-0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cAMP, its analogues, and forskolin on lung fluid production by in vitro lung preparations from fetal guinea pigs.

Lungs from fetal guinea pigs (62 +/- 1 days of gestation) were supported in vitro for 3 h and fluid production was determined by a dye dilution method, based on Blue Dextran 2000. Twenty untreated lungs produced fluid at 1.41 +/- 0.22 mL.kg-1 body weight.h-1, with no significant changes during later hours. Treatments with analogues of cAMP, cAMP, or forskolin during the middle hour reduced production significantly. Dibutyryl cAMP at 10(-3) M produced reabsorption (117.8 +/- 13.6% reduction, p less than 0.001, n = 10); at 10(-4) M it reduced production (77.3 +/- 11.0% fall, p less than 0.001, n = 10). 8-Bromo-cAMP appeared more effective; at 10(-4) M it caused slight reabsorption (109.0 +/- 8.9% reduction, p less than 0.001, n = 6) and at lower concentrations it decreased production (at 10(-6) M, 67.6 +/- 9.6% fall, p less than 0.001, n = 6; at 10(-7) M, 40.0 +/- 14.3% fall, p less than 0.001, n = 6). At high doses, cAMP itself produced similar effects (at 5 x 10(-3) M, 141.6 +/- 22.8% reduction, p less than 0.001, n = 6); at 10(-4) it was ineffective (n = 3). Forskolin at 10(-6) M induced the strongest reabsorptions seen (159.1 +/- 10.9% reduction, p less than 0.001, n = 6); at lower concentrations it reduced production (at 10(-8) M, 73.8 +/- 5.5% fall, p less than 0.001, n = 6; at 10(-9) M, 29.2 +/- 9.2% fall, p less than 0.05, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of exogenous 11-ketotestosterone, testosterone, and cyproterone acetate on prespawning and parental care behaviors of male bluegill.

This study investigated the effect of exogenous 11-ketotestosterone (11KT) and testosterone (T), two important androgens involved in fish reproduction, as well as cyproterone acetate (CYA), an antiandrogen, on behaviors of male bluegill (Lepomis macrochirus) during prespawning and parental care periods. Parental male bluegill build nests in colonies, spawn with females, and then remain alone on these nests to provide care for their developing offspring. Implantation of pellets containing either 11KT or T did not induce parental males to build nests in early spring, before the onset of natural spawning, or during late spring and early summer, when the reproductive season was in progress. Treatment with pellets containing CYA inhibited reproduction in male bluegill and these fish did not spawn for the remainder of the reproductive season. The release of appropriate steroids from pellets to the serum was confirmed in all cases by a radioimmunoassay. Interestingly, nesting males that spawned successfully and were implanted with 11KT at the onset of the parental care phase displayed increased rim-circling, a behavior characteristic of the late prespawning period. Treatment with T or CYA, however, did not induce such behaviors in nesting bluegill. Finally, none of these treatments significantly affected antipredator aggressive behavior displayed by nesting males. Our results suggest that parental care behaviors in male bluegill are most likely expressed through nonandrogenic mechanisms.

Serum 11-ketotestosterone and testosterone concentrations associated with reproduction in male bluegill (Lepomis macrochirus: Centrarchidae).

Male bluegill (Lepomis macrochirus) display a complex reproductive behavior involving two alternative life history pathways: delay of sexual maturation to become "parentals" or precocious maturation as "cuckolders." The purpose of our study was to investigate the association of two androgens, 11-ketotestosterone (11KT) and testosterone (T), with reproduction in these two types of males. Radioimmunoassay techniques were used to measure daily levels of the two androgens in the blood serum of parental male bluegill captured during the prespawning, spawning, and nesting periods throughout the reproductive season. Dramatic changes in the levels of 11KT and T were observed among parental males during these periods. Peaks occurred at the onset of spawning activity during each breeding bout. Compared to spawning parental males, spawning cuckolder males had significantly lower serum levels of 11KT. In contrast, the serum levels of T among parental and cuckolder males were not significantly different. These findings suggest that the elevated levels of 11KT are associated with the behaviors displayed by spawning parental males. The levels of T, however, seem to be associated with the occurrence of a phenomenon common to both parental and cuckolder males, such as development of gonads and/or spermiation.