Biomedical subjects

Maud Frieden

Publications and source records attributed to Maud Frieden.

6 recordsLinked to original sources

PubMed2003

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering, sore-operated Ca2+ entry, and Ca2+ store refilling.

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.

Biological Transport↗

PubMed2003

Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation.

In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.

Buffers↗

PubMed2003

Measurements of the free luminal ER Ca(2+) concentration with targeted "cameleon" fluorescent proteins.

The free ER Ca(2+) concentration, [Ca(2+)](ER), is a key parameter that determines both the spatio-temporal pattern of Ca(2+) signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca(2+) activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca(2+) signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca(2+) by optical means. The two main strategies used to date are Ca(2+)-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca(2+) indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded "cameleon" Ca(2+) indicators for ER Ca(2+) measurements.

Aequorin↗

PubMed2002

Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria.

To study the role of calreticulin in Ca(2+) homeostasis and apoptosis, we generated cells inducible for full-length or truncated calreticulin and measured Ca(2+) signals within the cytosol, the endoplasmic reticulum (ER), and mitochondria with "cameleon" indicators. Induction of calreticulin increased the free Ca(2+) concentration within the ER lumen, [Ca(2+)](ER), from 306 +/- 31 to 595 +/- 53 microm, and doubled the rate of ER refilling. [Ca(2+)](ER) remained elevated in the presence of thapsigargin, an inhibitor of SERCA-type Ca(2+) ATPases. Under these conditions, store-operated Ca(2+) influx appeared inhibited but could be reactivated by decreasing [Ca(2+)](ER) with the low affinity Ca(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. In contrast, [Ca(2+)](ER) decreased much faster during stimulation with carbachol. The larger ER release was associated with a larger cytosolic Ca(2+) response and, surprisingly, with a shorter mitochondrial Ca(2+) response. The reduced mitochondrial signal was not associated with visible morphological alterations of mitochondria or with disruption of the contacts between mitochondria and the ER but correlated with a reduced mitochondrial membrane potential. Altered ER and mitochondrial Ca(2+) responses were also observed in cells expressing an N-truncated calreticulin but not in cells overexpressing calnexin, a P-domain containing chaperone, indicating that the effects were mediated by the unique C-domain of calreticulin. In conclusion, calreticulin overexpression increases Ca(2+) fluxes across the ER but decreases mitochondrial Ca(2+) and membrane potential. The increased Ca(2+) turnover between the two organelles might damage mitochondria, accounting for the increased susceptibility of cells expressing high levels of calreticulin to apoptotic stimuli.

Apoptosis↗

PubMed2002

Subplasmalemmal endoplasmic reticulum controls K(Ca) channel activity upon stimulation with a moderate histamine concentration in a human umbilical vein endothelial cell line.

This study was designed to elucidate the role of the subplasmalemmal endoplasmic reticulum (sER) in autacoid-induced stimulation of Ca(2+)-dependent K(+) channels in the umbilical vein endothelial cell-derived cell line EA.hy926. Cells were transfected with the Ca(2+) probe cameleon targeted to the ER for visualization of the ER network. A patch pipette was then placed close to or far (> 5 microm away) from the sER, single channel recordings (patch clamp technique) were monitored simultaneously with measurements of either ER Ca(2+) concentration (using the Ca(2+) probe Cam4-ER) or cytosolic free Ca(2+) concentration ([Ca(2+)](i); using fura-2) using a deconvolution imaging device. A voltage-dependent, large conductance Ca(2+)-dependent K(+) channel (BK(Ca); single channel conductance (gamma), 250 pS) was found. At membrane potentials of +40 and -40 mV, the EC(50) for Ca(2+) was 2.7 and 49.7 microM, respectively. In the vicinity of the sER, the BK(Ca) channel activity induced by 10 microM histamine was 32 times higher (open probability (P(o)) = 0.083 +/- 0.026) than in areas away from the sER (P(o) = 0.0026 +/- 0.002). However, at supramaximal histamine stimulation (100 microM), BK(Ca) channel activation was similar in patches in the vicinity of or away from the sER (P(o) = 0.18 +/- 0.09 and 0.25 +/- 0.07, respectively). In contrast to BK(Ca) channel activity, ER Ca(2+) depletion (Cam4-ER) and elevation of [Ca(2+)](i) in response to 10 and 100 microM histamine were not influenced by the pipette position. We conclude that in endothelial cells, the activation of BK(Ca) channels in response to moderate histamine concentration essentially depends on the proximity of the sER domains to the mouth of this K(+) channel. These findings further support our concept of the subplasmalemmal Ca(2+) control unit (SCCU) and add the local activation of Ca(2+)-activated K(+)-channels to the function of the SCCU.

Calcium↗

PubMed2002

Tissue-specific expression of human lipoprotein lipase in the vascular system affects vascular reactivity in transgenic mice.

1. The role of smooth muscle-derived lipoprotein lipase (LPL) that translocates to the endothelium surface on vascular dysfunction during atherogenesis is unclear. Thus, the role of vascular LPL on blood vessel reactivity was assessed in transgenic mice that specifically express human LPL in the circulatory system. 2. Aortic free fatty acids (FFAs) were increased by 69% in the transgenic mice expressing human LPL in aortic smooth muscle cells (L2LPL) compared with their non-transgenic littermates (L2). 3. Contractility to KCl was increased by 33% in aortae of L2LPL mice. Maximal contraction to phenylephrine (PE) was comparable in L2 and L2LPL animals, while the frequency of tonus oscillation to PE increased by 104% in L2LPL mice. 4. In L2LPL animals, *NO mediated relaxation to acetylcholine (ACh) and ATP was reduced by 47 and 32%, respectively. In contrast, endothelium-independent relaxation to sodium nitroprusside (SNP) was not different in both groups tested. 5. ATP-initiated Ca(2+) elevation that triggers *NO formation was increased by 41% in single aortic endothelial cells freshly isolated from L2LPL animals. 6. In aortae from L2LPL mice an increased *O(2)(-) release occurred that was normalized by removing the endothelium and by the NAD(P)H oxidase inhibitor DPI and the PKC inhibitor GF109203X. 7. The reduced ACh-induced relaxation in L2LPL animals was normalized in the presence of SOD, indicating that the reduced relaxation is due, at least in part, to enhanced *NO scavenging by *O(2)(-). 8. These data suggest that despite normal lipoprotein levels increased LPL-mediated FFAs loading initiates vascular dysfunction via PKC-mediated activation of endothelial NAD(P)H oxidase. Thus, vascular LPL activity might represent a primary risk factor for atherosclerosis independently from cholesterol/LDL levels.

Acetylcholine↗