Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Abstract

Endothelium-mediated vasodilation is specifically enhanced in uterine circulation during pregnancy, and production of nitric oxide (NO) is increased in response to a wide array of agonists. Uterine artery endothelial cells from nonpregnant (NP-UAECs) or pregnant (P-UAECs) ewes maintained in culture still show a pregnancy-enhanced difference in ATP-stimulated endothelial NO synthase (eNOS; official symbol NOS3) activation, even though NOS3 protein, purinergic receptors, and associated cell signaling proteins are expressed at equal levels. We have also shown that the pregnancy-enhanced endothelial cell NO response to ATP requires an enhanced and sustained capacitative entry phase that is likely mediated via canonical transient receptor potential protein/inositol 1,4,5-trisphosphate receptor type 2 interaction. In this study, we now show by simultaneous video imaging of individual Fura-2-loaded cells that the pregnancy-enhanced capacitative entry phase is not continuous and equal in all cells, but is in fact mediated as a series of periodic [Ca(2+)](i) bursts within individual cells. Not only does pregnancy increase the number of bursts over a longer time period in individual cells, but also a greater proportion of cells exhibit this burst activity, and at high cell density this occurs in a synchronous manner. The mediator of cell synchronization is connexin 43 (Cx43) gap junctions because 1) Cx43 is readily detectable by Western blot analysis in UAECs, whereas Cx40 and Cx37 are weakly detected or absent, and 2) pregnancy-specific enhancement of [Ca(2+)](i) bursts by ATP is blocked by inhibitory loop peptides selective to Cx43 ((43,37)GAP27) but not by a scrambled control peptide or (40)GAP27 or (40,37)GAP26 peptides, which are specific to Cx40 or Cx37. The relationship between Ca(2+) bursts and NOS3 activation is further established by the finding that (43,37)GAP27 inhibits ATP-stimulated NOS3 activation but has no effect on cell mitogenesis. We conclude that it is pregnancy-enhanced gap junction communication between cells that underlies pregnancy enhancement of capacitative entry via TRPC3 and, in turn, NOS3 activation. Such improved gap junction function allows greater and more sustained [Ca(2+)](i) responses to agents such as ATP within a single cell, as well as the additional recruitment of greater numbers of cells to the response in a coordinated and synchronous manner to support enhanced NO production.

The loss of sustained Ca2+signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy

“…All standard culture media used for cell isolation or culture (MEM or M199) routinely contained 1% penicillin-streptomycin solution (Life Technologies) and 4 g/ml gentamycin (Life Technologies). The established methods of dual imaging of [Ca 2ϩ ]i response and simultaneous NO production utilized in intact UA Endo and the [Ca 2ϩ ]i imaging alone utilized in UAEC (25,26) were used in this study on the intact UV Endo and the cultured HUVEC from control and PE subjects.…”

“…These cells were grown to 95% confluence and trypsinized for freezing in media with 10% DMSO in 35 cryovials at passage 3. Individual cell preparations were independently validated by Western blot before use by growing to confluence in a 35-mm dish, lysed as below (see Western Blot Analysis), and analyzed for consistent expression of eNOS, ve-cadherin, and VEGFR2 as endothelial markers, as well as Cx43 necessary for Ca 2ϩ bursting (26) and Hsp90 as normalization control. Levels of these proteins in HUVEC preparations varied Ͻ10% within each group or between control and PE groups, with no significant difference observed (data not shown).…”

“…Solubilized protein was quantified using the BCA assay before separation of proteins on 7.5% gels with 10 ug protein/lane and transfer of proteins to an Immobilon-P PVDF membrane. Western analyses were performed as described previously (19,26). Total eNOS was detected using antieNOS (1:1,000) and rabbit anti-mouse IgG HRP-conjugated F(ab=)2 secondary antibody (1:2,500).…”

“…Our own detailed studies in this area have shown that increased output of NO by uterine artery endothelium in response to ATP exceeds changes in eNOS expression and is causally related to enhanced endothelial cell Ca 2ϩ signaling in the form of repeated intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) bursts [recently termed "adaptive programming" (6, 7)]. In turn, studies of these cells in primary culture show the sustained [Ca 2ϩ ] i burst response is a form of pregnancy-enhanced capacitative Ca 2ϩ entry (CCE) (9) that is facilitated by a pregnancy-specific increase in connexin 43 (Cx43) gap junction communication between endothelial cells, so facilitating more cells to exhibit burst responses during pregnancy and more synchronous bursts between cells (25,26). The uterine artery is not alone in showing this pregnancyenhanced adaptation of Ca 2ϩ signaling; a similar pregnancyspecific increase in the earliest CCE phase of [Ca 2ϩ ] i response to G protein-coupled receptor (GPCR)-coupled phospholipase C (PLC)-␤ agonists has also been implicated in maternal human hand vein endothelial cells (HHVEC) (13), and similar CCE profiles are seen in human umbilical vein endothelial cells (HUVEC) of normal umbilical cords (18).…”

Impairment of IKCachannels contributes to uteroplacental endothelial dysfunction in rat diabetic pregnancy

“…A [Ca 2ϩ ] i rise in response to ACh or ATP stimulation of ECs of the maternal uterine vasculature is a critical step in generating both NO and EDHF (25,52). Experimental diabetes during rat pregnancy significantly attenuates ACh-induced [Ca 2ϩ ] i responses in ECs of rat uteroplacental arteries (24).…”