Pituitary gonadotrophs are a small fraction of the anterior pituitary population, yet they synthesize gonadotropins: luteinizing (LH) and follicle-stimulating (FSH), essential for gametogenesis and steroidogenesis. LH is secreted via a regulated pathway while FSH release is mostly constitutive and controlled by synthesis. Although gonadotrophs fire action potentials spontaneously, the intracellular Ca2+ rises produced do not influence secretion, which is mainly driven by Gonadotropin-Releasing Hormone (GnRH), a decapeptide synthesized in the hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation. GnRH binding to G-protein-coupled receptors triggers Ca2+ mobilization from InsP3-sensitive intracellular pools, generating the global Ca2+ elevations necessary for secretion. Ca2+ signaling responses to increasing (GnRH) vary in stereotyped fashion from subthreshold to baseline spiking (oscillatory), to biphasic (spike-oscillatory or spike-plateau). This progression varies somewhat in gonadotrophs from different species and biological preparations. Both baseline spiking and biphasic GnRH-induced Ca2+ signals control LH/FSH synthesis and exocytosis. Estradiol and testosterone regulate gonadotropin secretion through feedback mechanisms, while FSH synthesis and release are influenced by activin, inhibin, and follistatin. Adaptation to physiological events like the estrous cycle, involves changes in GnRH sensitivity and LH/FSH synthesis: in proestrus, estradiol feedback regulation abruptly changes from negative to positive, causing the pre-ovulatory LH surge. Similarly, when testosterone levels drop after orquiectomy the lack of negative feedback on pituitary and hypothalamus boosts both GnRH and LH secretion, gonadotrophs GnRH sensitivity increases, and Ca2+ signaling patterns change. In addition, gonadotrophs proliferate and grow. These plastic changes denote a more vigorous functional adaptation in response to an extraordinary functional demand.

Gonadotropin-releasing hormone (GnRH) binds to pituitary gonadotroph receptors and initiates [Ca(2+)](i) signals and gonadotropin secretion. Here, we recorded GnRH-induced Ca(2+) signals in acute pituitary slices from both intact and castrated male mice 15 and 45 days after orchiectomy (GnX). Cells responding with "noncanonical" sequences of Ca(2+) signaling to increasing GnRH concentrations ([GnRH]; oscillatory responses at a given [GnRH] and transient responses at both lower and higher concentrations) were augmented significantly in the castrated mice. Also, 15 days after GnX the number and size of gonadotrophs were augmented, confirming earlier anatomical studies. Hypertrophied gonadotrophs after 15 days after GnX tended to display GnRH-induced Ca(2+) responses of greater amplitude. Furthermore, median effective dose (ED50) for GnRH decreased from 0.17 nM (control) to ~0.07 nM after GnX, suggesting increased GnRH responsiveness of the gonadotroph population. The progression of Ca(2+) response patterns reported in control male rat gonadotrophs (oscillations declining and spike-plateau responses dominating at increasing [GnRH]) was less conspicuous in mouse gonadotrophs in situ. Also, GnX-induced alterations in rat gonadotrophs (persistence of Ca(2+) oscillations even at [GnRH] >100 nM) were not mirrored by mouse gonadotrophs in situ. Contrary to observations in intact and 15-day castrated mice, after 45 days of GnX the hump component diminished and oscillations were augmented with increasing [GnRH], but Ca(2+) response patterns of gonadotrophs in situ remained virtually unchanged in response to [GnRH]s >1 nM, suggesting dose discrimination failure at high [GnRH]s. This study underscores the notion that GnRH responsiveness and the effects of testosterone deficiency may not be equal in pituitary gonadotrophs across species.

Cell penetrating peptides (CPPs) are molecules capable of passing through biological membranes. This capacity has been used to deliver impermeable molecules into cells, such as drugs and DNA probes, among others. However, the internalization of these peptides lacks specificity: CPPs internalize indistinctly on different cell types. Two major approaches have been described to address this problem: I) targeting, in which a receptor-recognizing sequence is added to a CPP, and ii) activation, where a non-active form of the CPP is activated once it interacts with cell target components. These strategies result in multifunctional peptides (i.e., penetrate and target recognition) that increase the CPP’s length, the cost of synthesis and the likelihood to be degraded or become antigenic. In this work we describe the use of machine-learning methods to design short selective CPP; the reduction in size is accomplished by embedding two or more activities within a single CPP domain, hence we referred to these as moonlighting CPPs. We provide experimental evidence that these designed moonlighting peptides penetrate selectively in targeted cells and discuss areas of opportunity to improve in the design of these peptides.

For several years, cell culture techniques have been physiologically relevant to understand living organisms both structurally and functionally, aiming at preserving as carefully as possible the in vivo integrity and function of the cells. However, when studying cardiac cells, glass or plastic Petri dishes and culture-coated plates lack important cues that do not allow to maintain the desired phenotype, especially for primary cell culture. In this work, we show that microscaffolds made with polydimethylsiloxane (PDMS) enable modulating the stiffness of the surface of the culture substrate and this originates different patterns of adhesion, self-organization, and synchronized or propagated activity in the culture of chick embryonic cardiomyocytes. Thanks to the calcium imaging technique, we found that the substrate stiffness affected cardiomyocyte adhesion, as well as the calcium signal propagation in the formed tissue. The patterns of activity shown by the calcium fluorescence variations are reliable clues of the functional organization achieved by the cell layers. We found that PDMS substrates with a stiffness of 25 kPa did not allow the formation of cell layers and therefore the optimal propagation of the intracellular calcium signals, while softer PDMS substrates with Young’s modulus within the physiological in vivo reported range did permit synchronized and coordinated contractility and intracellular calcium activity. This type of methodology allows us to study phenomena such as arrhythmias. For example, the occurrence of synchronized activity or rotors that can initiate or maintain cardiac arrhythmias can be reproduced on different substrates for study, so that replacement tissues or patches can be better designed.

Carcinogenesis is a multistage process involving the dysregulation of multiple genes, proteins, and pathways that make any normal cell acquire a cancer cell phenotype. Therefore, it is no surprise that numerous ion channels could be involved in this process. Since their discovery and subsequent cloning, ion channels have been established as therapeutic targets in excitable cell pathologies (e.g., cardiac arrhythmias or epilepsy); however, their involvement in non-excitable cell pathologies is relatively recent. Among all ion channels, the voltage-gated potassium channels Kv10.1 have been established as a promising target in cancer treatment due to their high expression in tumoral tissues compared to low levels in healthy tissues.