Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Lung hypoplasia in newborn rabbits with a diaphragmatic hernia affects pulmonary ventilation but not perfusion

BackgroundA congenital diaphragmatic hernia (DH) can result in severe lung hypoplasia that increases the risk of morbidity and mortality after birth; however, little is known about the cardiorespiratory transition at birth.MethodsUsing phase-contrast X-ray imaging and angiography, we examined the cardiorespiratory transition at birth in rabbit kittens with DHs. Surgery was performed on pregnant New Zealand white rabbits (n=18) at 25 days' gestation to induce a left-sided DH. Kittens were delivered at 30 days' gestation, intubated, and ventilated to achieve a tidal volume (V t) of 8 ml/kg in control and 4 ml/kg in DH kittens while they were imaged.ResultsFunctional residual capacity (FRC) recruitment and V t in the hypoplastic left lung were markedly reduced, resulting in a disproportionate distribution of FRC into the right lung. Following lung aeration, relative pulmonary blood flow (PBF) increased equally in both lungs, and the increase in pulmonary venous return was similar in both control and DH kittens.ConclusionThese findings indicate that nonuniform lung hypoplasia caused by DH alters the distribution of ventilation away from hypoplastic and into normally grown lung regions. During transition, the increase in PBF and pulmonary venous return, which is vital for maintaining cardiac output, is not affected by lung hypoplasia.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

European online postgraduate educational programme in neonatology—the way forward?

The provision of specialist postgraduate training is increasingly challenging for the acute medical specialties. There are often small numbers of trainees and tutors in any one centre, and service commitments may limit attendance at educational activities. Online learning can provide high-quality education to trainees from large geographical areas. We report the outcomes of an experimental educational project which provided an online postgraduate programme in neonatology. Ninety trainees from 14 countries, primarily European, participated. Six educational modules in neonatal topics were delivered over a 1-year period, within a “Virtual Learning Environment”. Trainees were divided into multi-national groups; two online tutors supported each group. Analysis of online activity demonstrated that active participation was high initially (100%) but gradually declined to 46% in the final module; tutor participation followed a similar pattern. Eighty-six trainees were contactable at the end of the programme, and 67 (78%) completed an evaluation questionnaire. Of these, 92% reported that participation had “added value” to their training, attributable to the high-quality curriculum, the educational resources, collaborative networking and the sharing of best practice. Eleven (79%) tutors completed the questionnaire, with all reporting that participation was of educational value. The main limiting factor for trainees and tutors was insufficient time. This project confirms that multi-national online education in neonatology is feasible and transferable, but for this approach to be viable formal accreditation and protected time for both trainees and tutors are required