Antonio Scarpa

Regulation of cell magnesium

Magnesium is the second most abundant cation within mammalian cells, trailing only potassium. It is also in- dispensable in the cell for a score of reactions and func- tions: it activates a large number of enzymes either di- rectly or by forming complexes with and modifying substrates; it maintains proper conformations of nucleic acids and proteins; it regulates the operation of channels, receptors, and intracellular signalling molecules; it mod- ulates photosynthesis, oxidative phosphorylation, muscle contraction, and nerve excitability; it is indispensable for cell growth and for controlling cell cycle (1, 2). It is thus amazing that we know so little about the cellular homeostasis of Mg2+ and its control. During the past several decades, all the other major cellular cations have engrossed the interest of biological researchers and this has resulted in a detailed elucidation of the various mechanisms of cellular Ca2+, H’, K+, or Na+ homeostasis. In fact, the number of recent published observations for each of these cations is so staggering that it would put the writer in a quandary to write a small review not only on their cellular control but also on individual compo- nents, organelles, or enzymes involved in specific steps of this control. This is not the case for Mg2+, for which observations in the literature related to its cellular control are sparse, despite some encouraging recent progress. The reasons for this limited knowledge are several, but the most prominent reason is that the techniques available for measuring cell Mg2+ are a far cry from those available for measuring other cations. Compounding this deficiency is the fact that the free Mg2+ concentration in cells is high, several orders of magnitude greater than that of Ca2+ or H+. Because of this, even large changes in intra- cellular Mg2+ mobilization represent only a small fraction

Cytosolic Ca2+ and the regulation of secretion in parathyroid cells

(Parathyroid cell) Secretion cytosolic Ca2+ Quin‐2 Fura‐2 Receptor

A direct analysis of lamellar x-ray diffraction from hydrated oriented multilayers of fully functional sarcoplasmic reticulum

The profile structure of functional sarcoplasmic reticulum (SR) membranes was investigated by X-ray diffraction methods to a resolution of 10 A. The lamellar diffraction data from hydrated oriented multilayers of SR vesicles showed monotonically increasing widths for higher order lamellar reflections, indicative of simple lattice disorder within the multilayer. A generalized Patterson function analysis, previously developed for treating lamellar diffraction from lattice-disordered multilayers, was used to identify the autocorrelation function of the unit cell electron density profile. Subsequent deconvolution of this autocorrelation function provided the most probable unit cell electron density profile of the SR vesicle membrane pair. The resulting single membrane profile possesses marked asymmetry, suggesting that a major portion of the Ca++ -ATPase resides on the exterior of the vesicle. The electron density profile also suggests that the Ca++-dependent ATPase penetrates into the lipid hydrocarbon core of the SR membrane. Under conditions suitable for X-ray analysis, SR vesicles prepared as partially dehydrated oriented multilayers are shown to conserve most of their ATP-induced Ca++ uptake functionality, as monitored spectrophotometrically with the Ca++ indicator arsenazo III. This has been verified both in resuspensions of SR after centrifugation and slow partial dehydration, and directly in SR multilayers in a partially dehydrated state (20-30 percent water). Therefore, the profile structure of the SR membrane that we have determined may closely resemble that found in vivo.

Calcium and pyridine nucleotide interaction in mitochondrial membranes

Abstract The interaction of NADH with divalent cations was studied in model systems and in isolated rat liver mitochondria. The addition of Ca 2+ to solutions of NADH in methanol, but not in water, produces a large increase in fluorescence intensity and a shift in the emission spectrum to a shorter wavelength. The ratio of fluorescence intensity in the presence of Ca 2+ with respect to that in the absence of Ca 2+ is 1 in aqueous and 2.4 in methanol solutions. Other di- or trivalent cations produce a similar enhancement of NADH fluorescence in nonpolar solutions, except for Mn 2+ which quenches the fluorescence of NADH. The energy-dependent accumulation of Ca 2+ in mitochondria increases the NADH fluorescence in the presence of rotenone. This effect was absent when Mn 2+ was previously accumulated by mitochondria. With the NADH-linked substrates, but not with succinate and rotenone, the accumulation of large amounts of Ca 2+ in mitochondria causes a depression of respiration and a release of the accumulated Ca 2+ . The inhibition of the respiration is accompanied by an increased permeability and a depletion of the endogenous pyridine nucleotides. Both effects were not directly related to the concomitant mitochondrial swelling. On the basis of this data, the formation of a complex between calcium and NADH in the nonpolar region of the mitochondrial membrane is proposed. The physiological implications of this complex are discussed.

Subcellular fractions of smooth muscle: Isolation, substrate utilization and Ca++ transport by main pulmonary artery and mesenteric vein mitochondria

Abstract Mitochondria were prepared from bovine mesenteric vein and main pulmonary artery in the presence of bovine serum albumin. Standard preparations contained between 30 and 60% of mitochondrial protein. Rates of substrate oxidation were measured polarographically. Succinate was oxidized at rates of 117.1 ± 16 and 92.3 ± 13 nmol of (O 2 /min/mg of main pulmonary artery and mesenteric vein mitochondrial protein, respectively. Other substrates were oxidized less rapidly. The calcium content of vascular smooth muscle mitochondria prepared in the presence and absence of EDTA was found to be much higher than in mitochondria from all other sources. Initial velocities of respiration-supported calcium uptake were measured by dual-wavelength techniques. Rates were slow at 1 μ m Ca 2+ but increased almost linearly up to 25 μ m Ca 2+ . The maximal velocities (12 and 4 nmol of Ca 2+ /s/mg of main pulmonary artery and mesenteric vein mitochondrial protein, respectively) were observed at 75 μ m Ca 2+ , and the half-maximal transports occurred at 17 μ m . At all [Ca 2+ ]tested, the rate of Ca 2+ transport was lower by mesenteric vein than by main pulmonary artery mitochondria. Vascular smooth muscle mitochondria can tolerate as much as 210 nmol of Ca 2+ mg of mitochondrial protein without uncoupling. These findings are discussed in terms of physiological regulation of cytosolic calcium levels in smooth muscle.

Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes.

Perfused rat hearts release or accumulate approximately 10% of total Mg2+ content when stimulated with norepinephrine (NE) or carbachol, respectively. Collagenase-dispersed rat ventricular myocytes increase or decrease total cell Mg2+ by 1 mM within 5 minutes when stimulated with these same transmitters. Measurements of Mg2+ transport using 28Mg or atomic absorbance spectrophotometry indicate that the rate and the extent of both stimulated Mg2+ efflux and influx are independent of the concentration of extracellular Mg2+ (0 to 1.2 mM). Mg2+ release induced by NE is rapidly reversed by the addition of carbachol, and Mg2+ uptake induced by carbachol is reversed by NE. Decreasing extracellular Na+ or Ca2+ decreases or abolishes Mg2+ efflux from myocytes. Cd2+ or other Ca2+ channel blockers also inhibit Mg2+ efflux in the presence of a physiological concentration of extracellular Ca2+. Replacement of extracellular Ca2+ with Sr2+ or with Mn2+ decreases or abolishes both stimulated efflux and influx of Mg2+. Redistribution of 85Sr in myocytes and in the supernatant indicates that under those conditions Sr2+ is released or accumulated by NE or carbachol in a manner similar to that of Mg2+. Hence, at least in the case of Sr2+, the inhibition of Mg2+ fluxes can be explained by the transport of Sr2+ rather than Mg2+ through the transport(s) systems. By contrast, replacement of extracellular Ca2+ with Ba2+ inhibits stimulated Mg2+ uptake but not Mg2+ release. These results indicate that cardiac myocytes have a major pool of Mg2+ that can be rapidly mobilized upon hormonal stimulation. The net uptake and release of Mg2+ are quantitatively similar and appear to be independent of the extracellular Mg2+ concentrations but are affected, to various degrees, by the presence of other cellular or extracellular cations.

Physical properties of biological membranes determined by the fluorescence of the calcium ionophore A23187

Abstract Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca 2+ , Mg 2+ , and Mn 2+ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current. On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca 2+ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca 2+ , Mg 2+ , or Mn 2+ are detected. A23187 produces fluorescent transients under conditions of Ca 2+ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca 2+ content. Changes in A23187 fluorescence due to mitochondrial Ca 2+ accumulation are much smaller by comparison and fluorescence transients are not detected. Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca 2+ and Mg 2+ , and less than 2 nsec in the presence of excess EDTA. The present results are consistent with a model of ionophore-mediated cation transport in which free M 2+ binds with A23187 at the membrane surface to form the complex M(A23187) + . Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187) 2 , which carries the divalent cation through the membrane. These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.

Determinants of Salt Sensitivity in Black and White Normotensive and Hypertensive Women

Abstract—Salt sensitivity (SS) has been linked to human hypertension. We examined ethnic differences in the relation between SS; erythrocyte sodium (Na+i), calcium (Ca2+i), potassium (K+i), and magnesium (Mg2+i); and sodium pump activity in African-American (AA) and white women. In a crossover protocol, similar numbers of normotensive, hypertensive, AA, and white women were randomized to 7 days of a 20 meq/d and a >200 meq/d salt diet (n=199). After an overnight inpatient stay, group differences in supine blood pressure (BP), heart rate, erythrocyte cations, and sodium pump activity were measured. The prevalence of SS (53.5% vs 51%) and salt resistance (26.3% vs 30.0%) was similar in both races. Greater mean BP increase with salt loading was seen in AA vs white hypertensives but not between the normotensive women. In hypertensives, increase in mean arterial pressure was 12.6 vs 8.2 mm Hg in AAs vs whites, respectively (P <0.01), and for systolic BP, it was 23 vs 14.8 mm Hg (P <0.01). Higher Na+i and Ca2+i were noted in SS and salt-intermediate AA than in the corresponding white subjects. Na+i, Ca2+i, and the ratios of Na+i to K+i and of Ca2+i to Mg2+i were positively correlated with salt responsiveness in AA but not in white women. Sodium pump activity was similar between groups, although the change in maximal activity trended to vary inversely with SS in AA. In closely matched AA and white women, the prevalence of SS is similarly high in both races, although the magnitude of BP increase is greater in AA hypertensives. In AA but not in whites, SS is positively associated with Na+i, Ca2+i, and the ratios of Na+i to K+i and of Ca2+i to Mg2+i.

The internal pH of mast cell granules.

Mast cells, which are found in all anatomical structures of the body except the blood and lymph, possess one of the largest contents of the biogenic amines histamine and serotonin (S-hydroxytryptamine) found within mammalian cells [ 1,2]. These amines, along with large amounts of the anionjc mucopolysaccharide heparin and proteolytic enzymes, are stored in high concentration within the osmophilic storage granules of the mast cell [3,4]. Varying in size from 300-800 nm, the spherical mast cell granules can be easily viewed by light microscopy [5]. According to [4] histamine is synthesized from histidine in the cytosol of the mast cell via the enzyme histidine decarboxylase, however fluorescence microscopy has established that histamine is localized in high concentration only in the granules [5]. Although this evidence suggests active ~stamine accumulation into mast cell granules against an apparent concentration gradient, the precise mechanism by which histamine and serotonin are transported and stored is unknown. Considerable similarity exists between the mast cell granule, the chromaffm granule, and the platelet dense granule with respect to the existence of a high content of basic biogenic amines, the presence of large i& acidic molecules implicated in the storage complex, the presence of associated peptide hormones, and the release by a stimulus coupling mechanism [ 1,671. Chromaffin granules and platelet granules, which store large quantities of catecholamines and serotonin, respectively, are remarkable in that they both maintain a distinctly acidic intragranular pH of 5.5 even after isolation and resuspension at neutral pH [6-81. There is good evidence that this intravesicular acidic pH is generated by a H+-translocating ATPase within the granule membrane; the

Ionophore mediated equilibration of calcium ion gradients in fragmented-sarcoplasmic reticulum.

Vesicular fragments of sarcoplasmic reticulum (SR), isolated from skeletal muscle, rapidly accumulate Ca*+ [l-3] in the presence of adenosine triphosphate (ATP). Owing to its high specific activity, SR constitutes an outstanding system for studies on ion interactions with biological membranes [4,5] , The kinetic behavior of Ca” uptake and the simultaneous occurrence of ATP hydrolysis suggest that Ca2+ is transported into the inner space of the membrane vesicles [4]. The resulting concentration gradient is made energetically possible through hydrolysis of ATP [6] . It was also shown that SR has a significant cation binding capacity, in the absence of ATP [7, 81. Although these binding sites have an affinity for Ca’+ lower than that displayed by SR [9, lo] in the presence of ATP, it was proposed that part or all of the ATP-dependent Ca2+ accumulation may be due to simple binding of Ca2+ rather than resulting from ionic gradients [7,8]. This problem may be clarified by the use of ionophores. We have now found that low concentrations of X-537 A, a Ca2+ ionophorous antibiotic, when added to SR suspensions after the occurrence of Ca2+ accumulation, produce a total release of the previously accumulated Ca”. Our results are consistent with the equilibration of the Ca ionic gradients, mediated by the ionophore X-537 A.