Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.
Cabilla JP, Ronchetti SA, Nudler SI, Miler EA, Quinteros FA, Duvilanski BH. Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands. Am J Physiol Endocrinol Metab 296: E731-E737, 2009. First published January 13, 2009 doi:10.1152/ajpendo.90795.2008.-17-Estradiol (E 2 ) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E 2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing ␣ 1-and decreasing 1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E 2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E 2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E 2 is the main effector of these changes. Since the sGC ␣-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC ␣-subunit are expressed. Here we show for the first time that sGC␣ 2 and sGC␣2 inhibitory (␣2i) isoforms are expressed in this gland, but only sGC␣ 2i mRNA increased after E2 acute treatment. Finally, to test whether E 2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGC␣ 1, -1, and -␣ 2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland. estrogen; soluble guanylyl cyclase; inhibitory subunit THE MAIN ESTROGENIC HORMONE 17-estradiol (E 2 ) plays important regulatory roles in a broad variety of biological processes, acting mainly on reproductive tissues, bone, liver, pituitary, and brain (9, 25).Nitric oxide (NO) is a signaling molecule that freely diffuses across cellular membranes where it binds to its main intracellular receptor, soluble guanylyl cyclase (sGC). This enzyme catalyzes the formation of cGMP from GTP. Subsequently, targets of cGMP such as cGMP-dependent protein kinases, cyclic nucleotide phosphodiesterases, and cyclic nucleotidesensitive ion channels are activated to continue the signal transduction (15,18).sGC is an heterodimeric enzyme and is comprised of two subunits, ␣ and , of which four types exist (␣ 1 , ␣ 2,  1 , and  2 ). Both ␣-isoforms form a functional enzyme with the  1 -subunit, although the ␣ 1  1 is the most abundant and widely expressed heterodimer, showing the greater activity (12, 13). The ␣ 2 is expressed in a more restricted pattern: in human tissues, it is present mainly in spleen, placenta, brain, ...
17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.
The variance in synaptic delays among endplate potentials events (referred here as jitter) was measured to study the contribution of voltage dependent calcium channels to transmission synchronicity in neuromuscular synapses from wild type and alpha-1A knockout mice (i.e., lacking P/Q type calcium channels). Knockout synapses presented higher jitter values than wild type ones under a wide range of extracellular calcium concentration ([Ca2+]o) values. Accordingly, wild type synapses showed less synchronic neurotransmitter release when P/Q type calcium channels were partially blocked as well as under lower [Ca2+]o. In the knockout synapses, N-type calcium channels mediated neurotransmitter release in a more temporally precise way than the R-type ones. Our results suggest that the type of calcium channels mediating transmitter release influenced the degree of synaptic synchrony. Thus, these results provide insight on the mechanisms underlying several pathologies associated with P/Q type calcium channels.
MAPK phosphatases (MKP) downregulate the activity of mitogen-activated protein kinases (MAPK), such as ERK1/2, and modulate the processes regulated by these kinases. ERK1/2 participate in a wide range of processes including tissue-specific hormone-stimulated steroidogenesis. H295R cells are a suitable model for the study of human adrenal cortex functions, particularly steroid synthesis, and respond to angiotensin II (Ang II) triggering ERK1/2 phosphorylation in a transient fashion. MKP-3 dephosphorylates ERK1/2 and, as recently reported, forkhead box protein 1 (FOXO1). Here, we analyzed MKP-3 expression in H295R cells and its putative regulation by Ang II. Results showed the expression of MKP-3 full length (L) and a short splice variant (S), and the upregulation of both isoforms by Ang II. L and S messenger and protein levels increased 30 min after Ang II stimulation and declined over the next 3 h, a temporal frame compatible with ERK1/2 dephosphorylation. In addition, FOXO1 activation is known to include its dephosphorylation and nuclear translocation. Therefore, we analyzed the effect of Ang II on FOXO1 modulation. Ang II induced FOXO1 transient phosphorylation and translocation and also the induction of p21, a FOXO1-dependent gene, whereas MKP-3 knock-down reduced both FOXO1 translocation and p21 induction. These data suggest that, through MKP-3, Ang II counteracts its own effects on ERK1/2 activity and also triggers the activation of FOXO-1 and the induction of cell cycle inhibitor p21. Taken together, the current findings reveal the participation of MKP-3 not only in turn-off but also in turn-on signals which control important cellular processes.
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