RESUMEN
Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (-30%) and Imax (-21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
Asunto(s)
Células Cromafines/metabolismo , Gránulos Cromafines/metabolismo , Epinefrina/metabolismo , Norepinefrina/metabolismo , Animales , Exocitosis/fisiología , Masculino , Ratones , Ratones Noqueados , Feniletanolamina N-Metiltransferasa/deficiencia , Feniletanolamina N-Metiltransferasa/genéticaRESUMEN
OBJECTIVE: It has been suggested that Ca entry through store-operated Ca channels (SOCs) is regulated by a dynamic interplay between the endoplasmic reticulum Ca stores and the mitochondria. These relationships drive the activation and inactivation of SOCs, yet it remains unclear whether this regulation of SOCs by mitochondria is altered in the aorta of spontaneously hypertensive rats (SHRs). METHODS: We performed a thorough study of the mitochondrial membrane potential, the ability of mitochondria to deal with cytosolic Ca, capacitative Ca entry (CCE), and stromal interaction molecule 1 (STIM1) and calcium release-activated calcium modulator 1 (orai1) protein expression, as well as the contractile capacity of aortic rings, in normotensive Wistar Kyoto rats (WKYs) and SHRs. RESULTS: Changes were observed in aortic tissue and cultured vascular smooth muscle cells isolated from SHRs relative to WKYs, including more depolarized mitochondria, stronger CCE upon the addition of Ca, larger cytosolic Ca transients (cytosolic Ca concentration) or aortic ring contraction elicited by endoplasmic reticulum depletion and a significant increase in STIM1 protein expression but not of orai1. CONCLUSION: These results suggest that the impaired Ca buffering capacity of partially depolarized mitochondria dysregulates CCE, leading to overfilling of the endoplasmic reticulum Ca store through enhanced STIM1/orai1 interactions and an increase in aorta contractions in SHRs. Thus, understanding the implications of the alterations to STIM1/orai1, and their relationship to mitochondria, may aid drug development and therapeutic strategies to treat hypertension, as well as its long-term sequelae in poorly controlled patients.
Asunto(s)
Aorta/fisiopatología , Canales de Calcio/metabolismo , Calcio/metabolismo , Hipertensión/fisiopatología , Animales , Aorta/metabolismo , Retículo Endoplásmico/metabolismo , Masculino , Mitocondrias/metabolismo , Músculo Liso Vascular/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.
Asunto(s)
Señalización del Calcio , Células Cromafines/metabolismo , Exocitosis , Médula Suprarrenal/citología , Médula Suprarrenal/embriología , Médula Suprarrenal/metabolismo , Animales , Catecolaminas/farmacología , Células Cultivadas , Células Cromafines/efectos de los fármacos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Ratas , Ratas WistarRESUMEN
Patterns of protein expression can be used to identify biomarkers of disease, prognosis or treatment response. Peripheral 5-HT2A and D3 receptors have been proposed as protein markers in schizophrenia. We investigated the possible parallel regulation of these candidate biomarkers in central nervous system (CNS) and peripheral blood cells by a comparative study of the effects of antipsychotic treatment on the expression of the receptors in both systems in rats. Acute (24 and 48 h) and subchronic (16 days) treatment of rats with olanzapine induced a significant decrease in 5-HT2A receptor density both in frontal cortex (Bmax=76.2%, 83.0% and 46.0% of control after 24 h, 48 h and 16 days of treatment, respectively; P<0.01) and blood platelets (Bmax approximately 55% of control at all times measured; P<0.01), without any changes in receptor affinity. Furthermore, olanzapine induced redistribution in 5-HT2A-like immunoreactivity and time-dependent remodelling of synaptic circuits involved in the activity of pyramidal and GABAergic neurons in frontoparietal motor cortex of treated rats, as assessed by immunohistochemical studies. D3 receptor mRNA levels increased significantly by 52.5% (P<0.01) and 21.1% (P<0.05) in nucleus accumbens, and by 53.4% (P<0.05) and 91.7% (P<0.01) in lymphocytes, after acute (24 h and 48 h) treatment with olanzapine, returning to levels similar to control after subchronic treatment (16 days). In conclusion, we observed in rats after olanzapine treatment: (1) parallelism in the regulation of 5-HT2A receptors in frontal cortex and in blood platelets; (2) parallelism in the regulation of D3 mRNA levels in nucleus accumbens and lymphocytes. These results endorse the interest in future studies aimed at validating these receptors as candidate biomarkers in schizophrenia.
Asunto(s)
Células Sanguíneas/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Receptor de Serotonina 5-HT2A/metabolismo , Receptores de Dopamina D3/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Animales , Benzodiazepinas/farmacología , Northern Blotting/métodos , Sistema Nervioso Central/metabolismo , Antagonistas de Dopamina/farmacocinética , Haloperidol/farmacocinética , Ketanserina/farmacocinética , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Olanzapina , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A/genética , Receptores de Dopamina D3/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Antagonistas de la Serotonina/farmacocinética , Tritio/farmacocinéticaRESUMEN
A series of 3-aminoethyl-1-tetralones, conformationally constrained higher homologues of haloperidol (standard for typical antipsychotic profile), have been obtained by a four-step route from valerolactone. Their binding affinities at dopamine D(2) and serotonin 5-HT2A and 5-HT2C receptors were determined, showing in some cases an atypical antipsychotic profile.