Uterine secretions are crucial for conceptus development in mammals. This is especially important for species that undergo extended preimplantation development, like cattle and other ungulates. The present study examined cooperative interactions for epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) on the proliferation of the bovine trophoblast cell line CT1 and bovine embryo development. Proliferation of CT1 cells increased after supplementation of the culture medium with 10ngmL EGF, 10ngmL FGF2 or 50ngmL IGF1, as well as with any combination of two factors. Greater increases in CT1 cell proliferation were detected when the growth medium was supplemented with all three factors. Supplementing the culture medium with individual or multiple factors during bovine embryo culture resulted in several positive outcomes, including increased blastocyst development, expansion, and hatching to varying degrees depending on the particular factor or combination of factors. Supplementation of the culture medium with all three factors increased embryonic trophoblast cell numbers on Day 8, as well as hatching rates and blastocyst diameter on Day 12 after fertilisation. Western blot analyses and the use of pharmacological inhibitors suggest that EGF and IGF1 affect CT1 proliferation by activating mitogen-activated protein kinase 3/1, whereas FGF2 activates AKT. In conclusion, the findings of the present study indicate that there are cooperative interactions among EGF, FGF2 and IGF1 that enhance trophoblast cell development during early embryogenesis.
We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (g9+) and electrogenic Na+/HCOj cotransport contributes importantly to regulation of intracellular pH (pH) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pH1 in the presence, but not absence, of HCO-, consistent with voltage-dependent HCO-influx. In the absence of HCO-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pH1-induced changes in gK+. By contrast, in the presence of HCO-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCOj cotransport. Moreover, the rate of p11 and potential difference recovery was severalfold greater in the presence as compared with the absence of HCO3T. Finally, continuous exposure to 10% CO2 in the presence ofHCO-produced intracellular acidification, and the rate of pH, recovery from intracellular acidosis was inhibited by Ba2+, which blocks p11-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO-cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pH, through effects on electrogenic Na+/HCOj cotransport. contrast to Na+/H' and CI-/HCO exchange, Na+/HCOj cotransport has been described in a limited number of cell types (15-19) and little is known regarding its regulation. Unlike Na+/H' and Cl-/HCO exchange, which are electroneutral, Na+/HCO5 cotransport in hepatocytes appears to carry net negative charge with each transport cycle (10-13) and, by analogy with similar mechanisms in other epithelia (16,17), is presumed to have a stoichiometry of two or three HC03 to each Na'. Thus, changes in transmembrane electrical potential difference (PD) might influence HCO transport and contribute to regulation of pH1.Hepatocellular PD is known to be strongly influenced by pHi such that intracellular acidification inhibits membrane K+ conductance (g9+) and depolarizes the membrane (20)(21)(22). This suggests that pH1-modulated changes in PD (mediated via pH-sensitive gK+) could alter the driving forces for electrogenic HCO influx, constituting a feedback mechanism for regulation of membrane HCO transport and pHi to meet changing metabolic demands. The model, depicted in Fig. 1, predicts that primary changes in transport or metabolism that lower pHi would decrease gK+ (step 1). The resulting membrane depolarization (step 2) would increase net HC03 influx via Na+/HCOj cotransport (step 3), thus buffering excess H+ and restoring pH;, gK+, and PD toward basal levels (step 4). The purpose of these studies was to test the possibility that such a func...
Recent studies in hepatocytes indicate that Na(+)-coupled HCO3- transport contributes importantly to regulation of intracellular pH and membrane HCO3- transport. However, the direction of net coupled Na+ and HCO3- movement and the effect of HCO3- on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3- on Na+ influx and turnover were measured in primary rat hepatocyte cultures with 22Na+, and [Na+]i was measured in single hepatocytes using the Na(+)-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na(+)-dependent or ouabain-suppressible 86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]i. In hepatocyte monolayers, HCO3- increased 22Na+ entry and turnover rates by 50-65%, without measurably altering 22Na+ pool size or cell volume, and HCO3- also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3- produced an abrupt and sustained rise in [Na+]i from approximately 8 to 12 mM. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased congruent to 2.5-fold in the presence of HCO3-, and the rise in [Na+]i produced by inhibition of the Na+/K+ pump was similarly increased congruent to 2.5-fold in the presence of HCO3-. In intact perfused rat liver, HCO3- increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3- movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3- to maintain [Na+]i within the physiologic range.
Na(+)-coupled HCO3- transport has been demonstrated in the basolateral membrane of hepatocytes, but there is uncertainty regarding its stoichiometry or capacity compared with other mechanisms of H(+)-HCO3- transport. After preincubation in medium free of Na+, either in the presence or absence of HCO3(-)-CO2, rat hepatocytes in primary culture were reexposed to Na+ or HCO3(-)-CO2 alone or in combination. Transporter electrogenicity was assessed by measuring membrane potential difference (PD), and the relative capacities of Na(+)-coupled HCO3- transport, Cl(-)-HCO3- exchange, and Na(+)-H+ exchange were assessed by measuring the magnitude and rate of change of intracellular pH (pHi) using BCECF. In the absence of Na+, exposure to HCO3- alone had no consistent effect on PD or pHi. In the absence of HCO3-, reexposure to Na+ depolarized cells by 3 +/- 1 mV and caused an amiloride-inhibitable increase in pHi of 0.031 +/- 0.02 units/min. In the presence of HCO3-, reexposure to Na+ hyperpolarized cells by -14 +/- 5 mV and increased pHi at a rate of 0.133 +/- 0.11 units/min; both the hyperpolarization and alkalinization were inhibited by SITS but unaffected by amiloride. These changes in PD indicate that Na(+)-coupled HCO3- transport is electrogenic, consistent with coupling of more than one HCO3- to each Na+. Furthermore, SITS-inhibitable Na(+)-dependent alkalinization exceeds amiloride-inhibitable Na(+)-dependent alkalinization by an order of magnitude, suggesting that the transport capacity of Na(+)-coupled HCO3- transport exceeds that of Na(+)-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)
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