Impairment of IKCa channels contributes to uteroplacental endothelial dysfunction in rat diabetic pregnancy.

Diabetes in rat pregnancy is associated with impaired vasodilation of the maternal uteroplacental vasculature. In the present study, we explored the role of endothelial cell (EC) Ca(2+)-activated K(+) channels of small conductance (SKCa channels) and intermediate conductance (IKCa channels) in diabetes-induced uterine vascular dysfunction. Diabetes was induced by injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with citrate buffer. Changes in smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and vasodilation induced by SKCa/IKCa channel activators were studied in uteroplacental arteries of control and diabetic rats. The impact of diabetes on SKCa- and IKCa-mediated currents was explored in freshly dissociated ECs. NS309 evoked a potent vasodilation that was effectively inhibited by TRAM-34 but not by apamin. NS309-induced smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and dilator responses were significantly diminished by diabetes; N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA)-evoked responses were not affected. Ca(2+)-activated ion currents in ECs were insensitive to paxilline, markedly inhibited by charybdotoxin (ChTX), and diminished by apamin. NS309-induced EC currents were generated mostly due to activation of ChTX-sensitive channels. Maternal diabetes resulted in a significant reduction in ChTX-sensitive currents with no effect on apamin-sensitive or CyPPA-induced currents. We concluded that IKCa channels play a prevalent role over SKCa channels in the generation of endothelial K(+) currents and vasodilation of uteroplacental arteries. Impaired function of IKCa channels importantly contributes to diabetes-induced uterine endothelial dysfunction. Therapeutic restoration of IKCa channel function may be a novel strategy for improvement of maternal uteroplacental blood flow in pregnancies complicated by diabetes.

Role of impaired endothelial cell Ca(2+) signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy.

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Downregulation of GluA2 AMPA receptor subunits reduces the dendritic arborization of developing spinal motoneurons.

AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca(2+) ions. Although Ca(2+)-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca(2+)-permeable AMPA receptors at E6 but the Ca(2+) permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca(2+) regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca(2+) permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development.

Pharmacological manipulation of GABA-driven activity in ovo disrupts the development of dendritic morphology but not the maturation of spinal cord network activity.

BACKGROUND: In the adult nervous system, GABA acts as a major inhibitory neurotransmitter; however, at early stages of neurodevelopment, GABA receptor activation leads to membrane depolarization and accumulation of [Ca2+]i. The role of excitatory GABAergic neurotransmission in the development of the nervous system is not fully understood. In this study, we investigated the role of excitatory GABA-driven activity in regulating the dendritic morphology and network function in the developing chicken spinal cord. RESULTS: Both bicuculline, a GABA receptor antagonist, and muscimol, a GABA agonist, inhibit the generation of spontaneous network activity in the isolated spinal cord at E8 or E10, indicating that altering GABA receptor activation disrupts the generation of spontaneous network activity in the chicken spinal cord. Treatment of chicken embryos with bicuculline or muscimol between E5 and E8 (or between E8 and E10), inhibits the dendritic outgrowth of motoneurons when compared to vehicle-treated embryos. The inhibitory effect of bicuculline or muscimol on the dendritic morphology of motoneurons was likely due to inhibition of GABA-driven network activity since a similar effect was also observed following reduction of network activity by Kir2.1 overexpression in the spinal cord. The inhibitory effect of bicuculline or muscimol was not caused by an adverse effect on cell survival. Surprisingly, chronic treatment of chicken embryos with bicuculline or muscimol has no effect on the shape and duration of the episodes of spontaneous activity, suggesting that maturation of network activity is not altered by disruption of the dendritic outgrowth of motoneurons. CONCLUSIONS: Taken together, these findings indicate that excitatory GABA receptor activation regulates the maturation of dendritic morphology in the developing spinal cord by an activity-dependent mechanism. However, inhibition of dendritic outgrowth caused by disruption of GABA-driven activity does not alter the maturation of spontaneous electrical activity generated by spinal cord networks, suggesting that compensatory mechanisms can reverse any adverse effect of dendritic morphology on network function.