Ricardo de Pascual

Endothelium-independent relaxation by 17-α-estradiol of pig coronary arteries

We have studied the effects of 17-α-estradiol, a non-estrogenic steroid, on pig coronary arteries contracted by K+, Ca2+ or serotonin. Experiments were performed on helicoidal strips and rings of left circumflex coronary arteries from freshly slaughtered white pigs and on helicoidal strips of rat thoracic aorta. The strips and rings were suspended inside a water-jacketed muscle chamber in an oxygenated Krebs solution at 37°C. From the initial K+-evoked contraction, 17-α-estradiol caused a relaxation with an IC50 (15 μM) which was in the range of the IC50s obtained for nitroglycerin (1.3 μM) and nicorandil (50 μM). Contractions evoked by Ca2+ were inhibited by 17-α-estradiol, but full blockade could not be achieved. Serotonin-evoked contractions were also blocked by 17-α-estradiol with an IC50 of 2.1 μM; 17-β-estradiol also inhibited the serotonin-evoked contractions. In the presence of 100 μM of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester, the relaxing properties of 17-α-estradiol in pig coronary arteries and rat thoracic aorta were unaffected, suggesting that endothelial NO release was unrelated to these effects. 17-α-Estradiol also relaxed denuded pig coronary artery strips, suggesting that other endothelial-derived relaxing factors were not involved in its vascular effects. The results are compatible with the idea that 17-α-estradiol causes relaxation of coronary vessels by acting directly on the cell membrane of smooth muscle cells; these effects seem to be unrelated to the genomic physiological effects of estrogens. These acute vasorelaxant effects can best be explained by blockade of voltage-dependent Ca2+ channels and the ensuing restriction of extracellular Ca2+ availability to the contractile machinery. This is in line with the recent hypothesis that estrogens behave as ‘endogenous Ca2+ channel antagonists’.

Voltage inactivation of Ca2+ entry and secretion associated with N‐ and P/Q‐type but not L‐type Ca2+ channels of bovine chromaffin cells

1 In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high‐voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2 The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+‐free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+‐free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. 3 This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura‐2‐loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. 4 A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. 5 The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N‐ and P/Q‐type Ca2+ channels. 6 This was strengthened by two additional findings: (i) nifedipine (3 μM), an L‐type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L‐type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7 In voltage‐clamped cells, switching the holding potential from ‐80 to ‐40 mV promoted the loss of 80 % of the whole‐cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. 8 Thus, it seems that the N‐ and P/Q‐subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L‐subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

Abstract Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 µM nifedipine (L-type channels), 21% sensitive to 1 µM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 µM ω-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 µM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.

Nicotine Promotes Initiation and Progression of KRAS-Induced Pancreatic Cancer via Gata6-Dependent Dedifferentiation of Acinar Cells in Mice

BACKGROUND & AIMS Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. METHODS We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. RESULTS Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program. CONCLUSIONS In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation.

Effects of ω-toxins on noradrenergic neurotransmission in beating guinea pig atria

The effects of four omega-toxins, known to block various subtypes of neuronal voltage-activated Ca2+ channels, on the beating guinea pig left atrium have been analyzed. Atria were suspended in oxygenated Krebs-bicarbonate solution at 32 degrees C and driven with electrical pulses delivered by a stimulator at 1 Hz, 1 ms, 4 V. A 10-fold increase of voltage caused a potent and rapid enhancement of the size of contractions (about 3- to 4-fold above basal), which reflects the release of endogenous noradrenaline from sympathetic nerve terminals. omega-Conotoxin MVIIC, omega-conotoxin MVIIA and omega-conotoxin GVIA inhibited the inotropic responses to 10 x V stimulation with IC50 values of 191, 44 and 20.4 nM, respectively. omega-Agatoxin IVA did not affect the contractile responses. The inotropic responses to exogenous noradrenaline were unaffected by the toxins. The potent blocking effects of omega-conotoxin GVIA were present even in conditions in which the release of noradrenaline was strongly facilitated by presynaptic alpha 2-adrenoceptor blockade by phenoxybenzamine. These effects were not reversed upon repeated washing of the tissue with toxin-free medium. In contrast, the blockade induced by omega-conotoxin MVIIC and omega-conotoxin MVIIA were fully reversed, with t1/2 of 13.5 and 31.2 min, respectively. omega-Conotoxin MVIIC (1 microM) protected against the irreversibility of the blockade induced by omega-conotoxin GVIA (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes.

1 The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (−) enantiomer R91154 on whole‐cell currents through Ca2+ channels, with 10 mM Ba2+ as charge carrier (IBa), have been studied in bovine and mouse voltage‐clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of −80 mV, at 10 s intervals had an average magnitude of 1 nA. 2 Lubeluzole and R91154 blocked the peak IBa of bovine chromaffin cells in a time and concentration‐dependent manner; their IC50s were 1.94 μM for lubeluzole and 2.54 μM for R91154. In a current‐voltage protocol, lubeluzole (3 μM) inhibited peak IBa at all test potentials. However, no obvious shifts of the I‐V curve were detected. 3 After 10 min exposure to 3 μM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered. 4 Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at −110 mV and 87% at −50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at −110 mV, decreased at −80 mV and did not occur at −50 mV. 5 Intracellular dialysis of bovine voltage‐clamped chromaffin cells with 3 μM lubeluzole caused neither blockade nor inactivation of IBa. The external application of 3 μM lubeluzole to those dialysed cells produced inhibition as well as inactivation of IBa. 6 The effects of lubeluzole (3 μM) on IBa in mouse chromaffin cells were similar to those in bovine chromaffin cells. At −80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late IBa (66%) as compared with peak IBa (46% after 10 min superfusion with lubeluzole). 7 In mouse chromaffin cells approximately half of the whole‐cell IBa was sensitive to 3 μM nifedipine (L‐type Ca2+ channels) and the other half to 3 μM ω‐conotoxin MVIIC (non‐L‐type Ca2+ channels). In ω‐conotoxin MVIIC‐treated cells, 3 μM lubeluzole caused little blockade and inactivation of IBa. However in nifedipine‐treated cells, lubeluzole caused a pronounced blockade and inactivation of IBa that reversed upon wash out of the compound. 8 The results are compatible with the idea that lubeluzole preferentially blocks non‐L‐types of voltage‐dependent Ca2+ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L‐type Ca2+ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca2+ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca2+ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.

Role of the Endoplasmic Reticulum and Mitochondria on Quantal Catecholamine Release from Chromaffin Cells of Control and Hypertensive Rats

Here, we present the first study on the effects of compounds that interfere with calcium (Ca2+) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K+ pulses triggered spike bursts of secretion by Ca2+ entry through Ca2+ channels. ER Ca2+ release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K+, CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca2+ handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K+ were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca2+ entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.

Single-Vesicle Catecholamine Release Has Greater Quantal Content and Faster Kinetics in Chromaffin Cells from Hypertensive, as Compared with Normotensive, Rats

In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K+ concentrations (K+) produced greater catecholamine release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced catecholamine secretory responses, we performed a single-vesicle release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K+. The secretory responses to ACh or K+ pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger catecholamine release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells

We established primary cultures of human pheochromocytoma chromaffin cells. We then tried to find what mechanism of their secretory apparatus could be altered to produce the massive release of catecholamines into the circulation and the subsequent hypertensive crisis observed in patients suffering this type of tumor. Their whole-cell Ca2+ channel currents could be pharmacologically separated into components similar to those found in normal human adrenal chromaffin cells: 20% L-type, 30% N-type, and 50% P/Q-type Ca2+ channels. However, modulation of the channels by exogenous or endogenous ATP and opioids, via a G-protein membrane-delimited pathway, was deeply altered; some cells having no modulation or very little modulation alternated with others having normal modulation. This may be the cause of the uncontrolled secretory response, measured amperometrically at the single-cell level. Some cells secreted for long time periods and were insensitive to nifedipine (L-type channel blocker) or to ω-conotoxin MVIIC (N/P/Q-type channel blocker), while others were highly sensitive to nifedipine and partially sensitive to ω-conotoxin MVIIC. Alteration of the autocrine/paracrine modulation of Ca2+ channels may lead to indiscriminate Ca2+ entry and exacerbate catecholamine release responses in human pheochromocytoma cells.

Selective activation of α7 nicotinic acetylcholine receptor (nAChRα7) inhibits muscular degeneration in mdx dystrophic mice

Amount evidence indicates that α7 nicotinic acetylcholine receptor (nAChRα7) activation reduces production of inflammatory mediators. This work aimed to verify the influence of endogenous nAChRα7 activation on the regulation of full-blown muscular inflammation in mdx mouse with Duchenne muscular dystrophy. We used mdx mice with 3 weeks-old at the height myonecrosis, and C57 nAChRα7(+/+) wild-type and nAChRα7(-/-) knockout mice with muscular injury induced with 60µL 0.5% bupivacaine (bp) in the gastrocnemius muscle. Pharmacological treatment included selective nAChRα7 agonist PNU282987 (0.3mg/kg and 1.0mg/kg) and the antagonist methyllycaconitine (MLA at 1.0mg/kg) injected intraperitoneally for 7 days. Selective nAChRα7 activation of mdx mice with PNU282987 reduced circulating levels of lactate dehydrogenase (LDH, a marker of cell death by necrosis) and the area of perivascular inflammatory infiltrate, and production of inflammatory mediators TNFα and metalloprotease MMP-9 activity. Conversely, PNU282987 treatment increased MMP-2 activity, an indication of muscular tissue remodeling associated with regeneration, in both mdx mice and WTα7 mice with bp-induced muscular lesion. Treatment with PNU282987 had no effect on α7KO, and MLA abolished the nAChRα7 agonist-induced anti-inflammatory effect in both mdx and WT. In conclusion, nAChRα7 activation inhibits muscular inflammation and activates tissue remodeling by increasing muscular regeneration. These effects were not accompanied with fibrosis and/or deposition of non-functional collagen. The nAChRα7 activation may be considered as a potential target for pharmacological strategies to reduce inflammation and activate mechanisms of muscular regeneration.