Calcium

Several factors during childhood and adolescence are thought to be associated with the development of proliferative benign breast diseases and breast cancer in adulthood. In order to identify them, the authors conducted an extensive review of the literature up to October 2022, searching for clinical studies, reports, and guidelines in English. A thorough Medline/Pubmed and Google scholar database research was performed, investigating the link between diet, exercise, age of menarche, body mass index, ionizing radiation exposure during childhood and adolescence, and proliferative breast diseases and breast cancer in adulthood. A list of keywords, including breast disorders, adolescence, childhood, and breast cancer was included in our search algorithm. Numerous studies concede that the development of breast disease in adulthood is influenced by various risk factors, whose influence begins during early childhood and adolescence.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the on state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danic...

To create muscle cell lines that conditionally differentiate in vitro we introduced a temperature-sensitive SV40 T antigen by retroviral infection into rat aortic smooth muscle cells (SMCs) and neonatal heart-derived cells. After G418 selection cell lines isolated were characterized at permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. [3H]Thymidine uptake showed tht progression through the cell cycle is greatly reduced at 39 degrees C. Cytoskeletal proteins, such as actins and vimentin did not change significantly after temperature shift, while the number of desmin-positive SMCs significantly increased when cells were switched to 39 degrees C. Heart-derived muscle cells showed sarcomeric myosin heavy chain reactivity only when grown at 39 degrees C. After thrombin stimulation intracellular calcium in both cell types increased severalfold in 39 degrees C-cells but not in 33 degrees C-cells. Whole cell patch-clamp recordings of SMCs and heart-derived cells reve...

The use of Mg2+-supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg2+supplementation on the regulation of intracellular calcium concentration ([Ca2+]i) and vascular function using an in vitro microvascular model. Ferret coronary arterioles (80–150 μm in diameter) were studied in a pressurized (40 mmHg) no-flow, normothermic (37°C) state. Simultaneous monitoring of internal luminal diameter and [Ca2+]iusing fura 2 were made with microscopic image analysis. The microvessels ( n = 6 each group) were divided into four groups according to the content of MgCl2(nominally 0, 1.2, 5.0, and 25.0 mM) in a hyperkalemic cardioplegic solution ([K+] 25.0 mM). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N2-5% CO2) containing each concentration of Mg2+([Mg2+]) and were then reoxygenated. Du...

We investigated if the magnitude of myogenic tone in the basilar artery of SHR differs from that in WKY and, if so, whether RhoA- or PKC-dependent mechanisms were involved. Myogenic tone was developed in response to stretch. Stretch-induced myogenic contraction was significantly greater in the SHR than WKY in the presence of external Ca2+. However, in the absence of external Ca2+, stretch did not evoke a myogenic tone. The [Ca2+]i-induced contraction was larger in SHR than WKY and the [Ca2+]i-force curve was significantly shifted to the left in SHR compared to WKY. Y-27632 significantly inhibited stretch-induced myogenic tone, but the inhibitory effect was larger in the SHR than WKY. However, PKC inhibitors had no significant effect on the myogenic tone. RhoA and PKCΕ were expressed at higher levels in the SHR compared to the WKY. RhoA and PKCα translocated from the cytosol to the cell membrane in response to stretch in both animals, but PKCΕ was translocated only in SHR. Our result...

The subcellular distribution of protein kinase C was directly imaged in single living smooth muscle cells using a new fluorescent protein kinase C probe. The probe localized prominently to perinuclear organelles and, to a lesser extent, to the cytosol and surface membrane. The perinuclear signal did not detectably change over the time observed. The ratio between the surface membrane intensity and that in the cytosol (R) was measured in parallel with cell shortening. In 1 mM extracellular Ca2+, the time to peak R and time to peak shortening were not significantly different. In Ca(2+)-free solution, no significant increase in the surface membrane/cytosol fluorescence ratio was observed with time, and shortening was inhibited. These results provide a new method for monitoring protein kinase C localization in living cells and directly demonstrate that protein kinase C moves in a Ca(2+)-dependent fashion from the cytosol to the surface membrane simultaneously with contraction.

The mechanisms by which prostaglandin F2 alpha (PGF2 alpha) can cause contractions at constant intracellular Ca2+ were investigated by the direct measurement of force from single saponin-permeabilized smooth muscle cells from the ferret aorta. The size of PGF2 alpha contractions did not change between pCa 9.0 and pCa 6.6. The remainder of the experiments were carried out at pCa 7.0. At pCa 7.0, PGF2 alpha (0.1-100 microM) induced sustained force in a dose-dependent manner, reaching a maximum (2.61 +/- 0.20 microN, n = 14) in 10 minutes. Both protein kinase C pseudosubstrate inhibitor (3 microM) and staurosporine (1 microM) significantly inhibited PGF2 alpha (100 microM)-induced contractions, but staurosporine was more effective. Staurosporine caused 88.8 +/- 13.3% inhibition, whereas protein kinase C pseudosubstrate inhibitor inhibited 62.3 +/- 9.6% of the PGF2 alpha-induced contraction. An inhibitor of type-1 and type-2A protein phosphatases, microcystin-LR, at a concentration of 1...

Little is known about the signaling pathways involved in alpha 2-adrenergic receptor-mediated contraction of vascular smooth muscle. In the present study, we measured intracellular Ca2+ ([Ca2+]i), myosin light chain (MLC) phosphorylation, and myofilament Ca2+ sensitivity during stimulation with the relatively selective alpha 2-agonist UK 14304. These effects were compared and contrasted with corresponding changes during depolarization by elevation of the [K+] in the bathing medium. These studies were performed using spiral strips of the rabbit saphenous vein, a tissue with a relatively high density of postsynaptic alpha 2-receptors. UK 14304 (10(-5) M) caused parallel changes in [Ca2+]i, MLC phosphorylation, and force consisting of an initial phasic, followed by a sustained steady-state response. The steady-state increase in [Ca2+]i, MLC phosphorylation, and force caused by UK 14304 in the presence of 2.5 mM extracellular Ca2+ were indistinguishable from those during 51 mM K+ depola...

Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled...

Background The hypermetabolic state induced by acute endotoxemia and malignant hyperthermia (MH) may be indistinguishable. The aims of this study were (1) to investigate the differences between MH and sepsis, (2) to determine whether acute endotoxemia can trigger MH, and (3) to establish the effects of dantrolene in these two disorders. Methods Three groups of swine were studied. All pigs were invasively monitored and initially anesthetized with nontriggering agents. A placebo MH-susceptible group (n = 5) received normal saline whereas the endotoxin groups (MH-susceptible, n = 6; MH-negative, n = 4) received intravenous endotoxin (250 microg/kg total) during 2.5 h. Halothane (1.5%) and succinylcholine (2-4 mg/kg) were then administered, followed by two doses of dantrolene (4 mg/kg total). Results Endotoxin infusion resulted in pulmonary hypertension and systemic hypotension in pigs with and without the MH mutation, but did not trigger MH. Halothane and succinylcholine triggered MH, ...

Objective: This study aimed to determine the prevalence of abnormal biochemical parameters among neonates with perinatal asphyxia in comparison to their non-asphyxiated controls.Methodology: This is a prospective case - control study involving 54 asphyxiated term neonates and 54 non-asphyxiated term babies at LAUTECH teaching hospital, Osogbo. Serum levels of Sodium, Bicarbonate, Chloride, Calcium and Potassium were determined daily for 72 hours in both groups using standard methods. The results were compared.Results: The overall prevalence of abnormal biochemical parameters namely hyponatraemia, hypocalcaemia, metabolic acidosis, hypochloraemia and hypokalaemia among the asphyxiated versus non-asphyxiated babies in the first 72 hours of life were 30.9% vs 19.8% (p < 0.020); 28.4% vs 4.9% (p < 0.000); 30.9% vs 3.1% (p < 0.0001); 27.2% vs 25.9% (p < 0.200) and 24.7% vs 3.1% (p < 0.070) respectively.Conclusion: Babies with hypoxic ischaemic encephalopathy stage III sign...

The mechanisms by which glutamatergic neurotransmitters modulate neuronal lipid metabolism are not well established. We have directly measured phospholipase A2 (PLA2) enzymic activity in cell-free extracts from cortical neuronal cultures from rat brain and have found that the PLA2 activity is up-regulated after cells are exposed to glutamate. Brief exposure to a calcium ionophore or phorbol 12-myristate 13-acetate (PMA) stably enhanced PLA2 activity. Down-regulation of protein kinase C activity partially blocked glutamate's effects. Two Ca(2+)-and pH-dependent forms of PLA2 were identified in cytosolic extracts. Activation of both forms of PLA2 was enhanced by prior exposure of the cultures to glutamate. One of the two forms had chromatographic characteristics on heparin-Sepharose, Mono Q and Superose 12 columns similar to the 100 kDa cytosolic PLA2 (cPLA2), and was recognized by an antibody raised to pig spleen cPLA2. The second form was similar in size to Group-I and -II PLA2s...