The adducin heterodimer is a protein affecting the assembly of the actin-based cytoskeleton. Point mutations in rat adducin alpha (F316Y) and beta (Q529R) subunits are involved in a form of rat primary hypertension (MHS) associated with faster kidney tubular ion transport. A role for adducin in human primary hypertension has also been suggested. By studying the interaction of actin with purified normal and mutated adducin in a cell-free system and the actin assembly in rat kidney epithelial cells (NRK-52E) transfected with mutated rat adducin cDNA, we show that the adducin isoforms differentially modulate: (a) actin assembly both in a cell-free system and within transfected cells; (b) topography of alpha V integrin together with focal contact proteins; and (c) Na-K pump activity at V(max) (faster with the mutated isoforms, 1281 +/- 90 vs 841 +/- 30 nmol K/h.mg pt., P < 0.0001). This co-modulation suggests a role for adducin in the constitutive capacity of the epithelia both to transport ions and to expose adhesion molecules. These findings may also lead to the understanding of the relation between adducin polymorphism and blood pressure and to the development of new approaches to the study of hypertension-associated organ damage.

It is known that cell density influences the maturation process of in vitro neuronal networks. Neuronal cultures plated with different cell densities differ in number of synapses per neuron and thus in single neuron synaptic transmission, which results in a density-dependent neuronal network activity. Although many authors provided detailed information about the effects of cell density on neuronal culture activity, a dedicated report of density and age influence on neuronal hippocampal culture activity has not yet been reported. Therefore, this work aims at providing reference data to researchers that set up an experimental study on hippocampal neuronal cultures, helping in planning and decoding the experiments. In this work, we analysed the effects of both neuronal density and culture age on functional attributes of maturing hippocampal cultures. We characterized the electrophysiological activity of neuronal cultures seeded at three different cell densities, recording their spontaneous electrical activity over maturation by means of MicroElectrode Arrays (MEAs). We had gather data from 86 independent hippocampal cultures to achieve solid statistic results, considering the high culture-to-culture variability. Network activity was evaluated in terms of simple spiking, burst and network burst features. We observed that electrical descriptors were characterized by a functional peak during maturation, followed by a stable phase (for sparse and medium density cultures) or by a decrease phase (for high dense neuronal cultures). Moreover, 900 cells/mm² cultures showed characteristics suitable for long lasting experiments (e.g. chronic effect of drug treatments) while 1800 cells/mm² cultures should be preferred for experiments that require intense electrical activity (e.g. to evaluate the effect of inhibitory molecules). Finally, cell cultures at 3600 cells/mm² are more appropriate for experiments in which time saving is relevant (e.g. drug screenings). These results are intended to be a reference for the planning of in vitro neurophysiological and neuropharmacological experiments with MEAs.

Protein kinase A (PKA) modulates several steps of synaptic transmission. However, the identification of the mediators of these effects is as yet incomplete. Synapsins are synaptic vesicle (SV)-associated phosphoproteins that represent the major presynaptic targets of PKA. We show that, in hippocampal neurons, cAMP-dependent pathways affect SV exocytosis and that this effect is primarily brought about through synapsin I phosphorylation. Phosphorylation by PKA, by promoting dissociation of synapsin I from SVs, enhances the rate of SV exocytosis on stimulation. This effect becomes relevant when neurons are challenged with sustained stimulation, because it appears to counteract synaptic depression and accelerate recovery from depression by fostering the supply of SVs from the reserve pool to the readily releasable pool. In contrast, synapsin phosphorylation appears to be dispensable for the effects of cAMP on the frequency and amplitude of spontaneous synaptic currents and on the amplitude of evoked synaptic currents. The modulation of depolarization-evoked SV exocytosis by PKA phosphorylation of synapsin I is primarily caused by calmodulin (CaM)-dependent activation of cAMP pathways rather than by direct activation of CaM kinases. These data define a hierarchical crosstalk between cAMP- and CaM-dependent cascades and point to synapsin as a major effector of PKA in the modulation of activity-dependent SV exocytosis.