Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Role of intracellular Ca2+ stores in smooth muscle contractions of the guinea pig vas deferens

Summary Guinea pig vas deferens was used as an animal model for alpha-1 adrenoceptor (₁-receptor) mediated contractions in human hyperplastic prostatic tissue. The selective ₁-receptor agonist, phenylephrine (PE), induced fully reversible, dose-dependent contractions antagonized by increasing concentrations of the ₁-receptor blockers prazosin (1–100 nM) and YM 617 (0.1–10 nM). Removal of extracellular Ca²⁺ reduced PE-evoked contractions in a time-dependent manner. Nifedipine (1–1000 nM), a blocker of voltage-dependent L-type Ca²⁺ channels (VDCC), inhibited the PE-induced response by up to 65%. Removal of extracellular Ca²⁺ abolished the ₁-agonist reactivity in a time-dependent fashion. To elucidate the participation of intracellular Ca²⁺ stores in ₁-receptor contractions, the tissue was pretreated with ryanodine (10 M) or thapsigargin (0.1 M), established inhibitors of Ca²⁺ release from intracellular pools. Both substances reduced the PE contractions by up to 80%. Nifedipine suppressed the remaining contractions completely. This provides evidence that Ca²⁺ influx through VDCC and Ca²⁺ release from intracellular stores contribute to ₁-receptor contractions in the guinea pig vas deferens and may be important in obstructive benign prostatic hyperplasia.     

Mobilization of intracellular calcium stores participates in the rise of [Ca2+]i and the toxic actions of the HIV coat protein GP120

The HIV envelope glycoprotein, GP120, increases intracellular Ca2+ concentration and induces degeneration of human and animal neurons in culture. Using patch-clamp recordings and Ca2+ imaging techniques, we have now examined the contribution of intracellular stores of calcium in the effects of GP120. We report that in rat hippocampal neuronal cultures, GP120 induces a dramatic and persistent increase in [Ca2+]i which is prevented by drugs that either deplete (caffeine, carbachol, thapsigargin) or block (dantrolene) Ca2+ release from intracellular stores. In contrast, N-methyl-d-aspartate (NMDA) receptors or voltage-dependent calcium channels do not participate in these effects, as: (i) the increase in [Ca2+]i was not affected by NMDA receptor antagonists or calcium channel blockers; and (ii) and GP120 did not generate any current in whole-cell recording. Dantrolene, a ryanodine stores inhibitor, also prevented neuronal death induced by GP120. Our results show that the GP120-induced rise in [Ca2+]i originates from intracellular calcium stores, and suggest that intracellular stores of calcium may play a determinant role in the pathological actions of GP120.     

Iron Stores of Breastfed Infants during the First Year of Life

The birth iron endowment provides iron for growth in the first months of life. We describe the iron endowment under conditions of low dietary iron supply. Subjects were infants participating in a trial of Vitamin D supplementation from 1 to 9 months. Infants were exclusively breastfed at enrollment but could receive complementary foods from 4 months but not formula. Plasma ferritin (PF) and transferrin receptor (TfR) were determined at 1, 2, 4, 5.5, 7.5, 9 and 12 months. At 1 month PF ranged from 38 to 752 µg/L and was only weakly related to maternal PF. PF declined subsequently and flattened out at 5.5 months. PF of females was significantly higher than PF of males except at 12 months. TfR increased with age and was inversely correlated with PF. PF and TfR tracked strongly until 9 months. Iron deficiency (PF < 10 µg/L) began to appear at 4 months and increased in frequency until 9 months. Infants with ID were born with low iron endowment. We concluded that the birth iron endowment is highly variable in size and a small endowment places infants at risk of iron deficiency before 6 months. Boys have smaller iron endowments and are at greater risk of iron deficiency than girls.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Use-dependent control of presynaptic calcium signalling at central synapses

Voltage-gated Ca(2+) channels activated by action potentials evoke Ca(2+) entry into presynaptic terminals thus briefly distorting the resting Ca(2+) concentration. When this happens, a number of processes are initiated to re-establish the Ca(2+) equilibrium. During the post-spike period, the increased Ca(2+) concentration could enhance the presynaptic Ca(2+) signalling. Some of the mechanisms contributing to presynaptic Ca(2+) dynamics involve endogenous Ca(2+) buffers, Ca(2+) stores, mitochondria, the sodium-calcium exchanger, extraterminal Ca(2+) depletion and presynaptic receptors. Additionally, subthreshold presynaptic depolarization has been proposed to have an effect on release of neurotransmitters through a mechanism involving changes in resting Ca(2+). Direct evidence for the role of any of these participants in shaping the presynaptic Ca(2+) dynamics comes from direct recordings of giant presynaptic terminals and from fluorescent Ca(2+) imaging of axonal boutons. Here, some of this evidence is presented and discussed.     

A Food-Derived Dietary Supplement Containing a Low Dose of Iron Improved Markers of Iron Status Among Nonanemic Iron-Deficient Women

Objective: Iron deficiency is the most common nutrient deficiency in the world. While deficiency can often be resolved through dietary supplementation with iron, adverse events are common and frequently preclude compliance. The objective of this study was to determine whether a food-derived dietary supplement containing a low dose of iron and nutrients that increase iron absorption could resolve iron deficiency with fewer adverse events than reported at higher doses.

Methods: A pilot clinical trial (NCT02683369) was conducted among premenopausal women with nonanemic iron deficiency that was verified by blood screening. Participants consumed a dietary supplement (Blood Builder®/Iron Response®) once daily for 8 weeks containing 26 mg of iron, vitamin C, folate, and other food-derived nutrients. Primary outcomes were markers of iron status (serum ferritin, hemoglobin, soluble transferrin receptor, total body iron stores) and secondary outcomes were self-reported fatigue and energy. All outcomes were assessed at baseline and 8 weeks. Adverse events were monitored with questionnaires, daily diaries, and contact with a physician. Dependent samples t test and Wilcoxon signed-rank test were used to analyze outcomes.

Results: Twenty-three participants enrolled in the study. Iron deficiency was resolved in the sample (mean serum ferritin: baseline = 13.9 μg/L, 8 weeks = 21.1 μg/L, p < 0.001). All other markers of iron status, fatigue, and energy also improved during the study (p < 0.04). No adverse events were reported. Conclusions: While larger and controlled studies are needed to confirm these findings, a food-derived dietary supplement with a low dose of iron and absorption-enhancing nutrients resolved iron deficiency and improved all other markers of iron status without any adverse events.     

Calculation algorithms alter the breath‐by‐breath gas exchange values when abrupt changes in ventilation occur

The automatic metabolic units calculate breath‐by‐breath gas exchange from the expiratory data only, applying an algorithm (‘expiration‐only’ algorithm) that neglects the changes in the lung gas stores. These last are theoretically taken into account by a recently proposed algorithm, based on an alternative view of the respiratory cycle (‘alternative respiratory cycle’ algorithm). The performance of the two algorithms was investigated where changes in the lung gas stores were induced by abrupt increases in ventilation above the physiological demand. Oxygen, carbon dioxide fractions and ventilatory flow were recorded at the mouth in 15 healthy subjects during quiet breathing and during 20‐s hyperventilation manoeuvres performed at 5‐min intervals in resting conditions. Oxygen uptakes and carbon dioxide exhalations were calculated throughout the acquisition periods by the two algorithms. Average ventilation amounted to 6·1 ± 1·4 l min^?1 during quiet breathing and increased to 41·8 ± 27·2 l min^?1 during the manoeuvres (P<0·01). During quiet breathing, the two algorithms provided overlapping gas exchange data and noise. Conversely, during hyperventilation, the ‘alternative respiratory cycle’ algorithm provided significantly lower gas exchange data as compared to the values yielded by the ‘expiration‐only’ algorithm. For the first breath of hyperventilation, the average values provided by the two algorithms amounted to 0·37 ± 0·34 l min^?1 versus 0·96 ± 0·73 l min^?1 for O₂ uptake and 0·45 ± 0·36 l min^?1 versus 0·80 ± 0·58 l min^?1 for exhaled CO₂ (P<0·001 for both). When abrupt increases in ventilation occurred, such as those arising from a deep breath, the ‘alternative respiratory cycle’ algorithm was able to halve the artefactual gas exchange values as compared to the ‘expiration‐only’ approach.