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The aim of this study was to evaluate cell diversity by considering how Ca2+ signaling has been adapted in skeletal muscle cell function. We characterized single C2C12 myoblasts through intracellular Ca2+ signaling kinetics after exposure to specific drugs and calcium blockers using fast fluorescence microspectrofluorimetry followed by ATP effect analysis, which confirmed the expression of functional purinergic adenosine and P2 receptors. Further, we found that glutamate sensitivity of C2C12 cells was mediated by ionotropic glutamate receptors; on the other hand, most cells were responsive to cyclopiazonic acid, which inhibits the sarco-endoplasmic reticulum Ca2+-ATPase pump. These results suggest that C2C12 cells possess functional L- and P/Q-type voltage-operated Ca2+ channels, ryanodine receptors and functional sarcoplasmic reticulum Ca2+ stores (typical for muscle cells), adenosine and P2 purinergic receptors, as well as ionotropic glutamate receptors. The evaluation of intracellular Ca2+ signaling is a promising approach towards a better understanding and control of the physiopathological properties of myogenic cells that could be used as a predictive factor in the selection of optimal cells for scaffold recellularization or for tissue engineered constructs used in stem cell therapy. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) consists of proteins, glycosaminoglycans and glycoproteins, that support the dynamic interactions between cells, including intercellular communication, cell attachment, cell differentiation, cell growth and migration. As such, the ECM represents an essential and very sensitive system within the tissue microenvironment that is involved in processes such as tissue regeneration and carcinogenesis. The aim of the present review is to evaluate its diversity through Ca2+ signaling and its role in muscle cell function. Here, we discuss some methodological approaches dissecting Ca2+ handling mechanisms in myogenic and non-myogenic cells, e.g. the importance of Ca2+ and calpains in muscle dystrophy. We also consider the reconstruction of skeletal muscle by colonization of decellularized ECM with muscle-derived cells isolated from skeletal muscle. Therefore, it is necessary to establish new methodological procedures based on Ca2+ signaling in skeletal muscle cells and their effect on ECM homeostasis, allowing the monitoring of skeletal muscle reconstruction and organ repair. [ABSTRACT FROM AUTHOR]

Salusin-α and -β were recently discovered as bioactive endogenous peptides. In the present study, we investigated the effects of chronic osmotic stimuli on salusin-β-like immunoreactivity (LI) in the rat hypothalamo-neurohypophyseal system. We examined the effects of salusin-β on synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and neurohypophyseal hormone release from both freshly dissociated SONs and neurohypophyses in rats. Immunohistochemical studies revealed that salusin-β-LI neurones and fibres were markedly increased in the SON and the magnocellular division of the paraventricular nucleus after chronic osmotic stimuli resulting from salt loading for 5 days and dehydration for 3 days. Salusin-β-LI fibres and varicosities in the internal zone of the median eminence and the neurohypophysis were also increased after osmotic stimuli. Whole-cell patch-clamp recordings from rat SON slice preparations showed that salusin-β did not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. In vitro hormone release studies showed that salusin-β evoked both arginine vasopressin (AVP) and oxytocin release from the neurohypophysis, but not the SON. In our hands, in the neurohypophysis, a significant release of AVP and oxytocin was observed only at concentrations from 100 nm and above of salusin-β. Low concentrations below 100 nm were ineffective both on AVP and oxytocin release. We also measured intracellular calcium ([Ca2+]i) increase induced by salusin-β on freshly-isolated single nerve terminals from the neurohypophysis devoid of pars intermedia. Furthermore, this salusin-β-induced [Ca2+]i increase was blocked in the presence of high voltage activated Ca2+channel blockers. Our results suggest that salusin-β may be involved in the regulation of body fluid balance by stimulating neurohypophyseal hormone release from nerve endings by an autocrine/paracrine mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ +Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors. [Copyright &y& Elsevier]

Oxytocin release from neurophypophysial terminals is particularly sensitive to inhibition by the µ-opioid receptor agonist, DAMGO. Because the R-type component of the neurophypophysial terminal Ca2+ current (ICa) mediates exclusively oxytocin release, we hypothesised that µ-opioids could preferentially inhibit oxytocin release by blocking this channel subtype. Whole-terminal recordings showed that DAMGO and the R-type selective blocker SNX-482 inhibit a similar ICa component. Measurements of [Ca2+]i levels and oxytocin release confirmed that the effects of DAMGO and SNX-482 are not additive. Finally, isolation of the R-type component and its associated rise in [Ca2+]i and oxytocin release allowed us to demonstrate the selective inhibition by DAMGO of this channel subtype. Thus, µ-opioid agonists modulate specifically oxytocin release in neurophypophysial terminals by selectively targeting R-type Ca2+ channels. Modulation of Ca2+ channel subtypes could be a general mechanism for drugs of abuse to regulate the release of specific neurotransmitters at central nervous system synapses. [ABSTRACT FROM AUTHOR]