No abstract

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.

Inositol 1,4,5-trisphosphate (InsP3) caused Ca release and tension development in rabbit main pulmonary artery smooth muscle permeabilized with saponin or digitonin. Both of these responses to single additions of uM) were repeatable and occurred in the presence of 0.0-1.9 mM free Mg +. Sustained contractions were induced by InsP3. The amount of Ca released by InsP3, measured with a Ca21-selective electrode, was also estimated to be sufficient to stimulate contraction in intact smooth muscle. Ca release was not influenced by inhibitors of mitochondrial oxidative phosphorylation. The uptake of Ca21 from the medium into the InsP3-sensitive pool was ATP-dependent. The present results support the hypothesis that, in smooth muscle, InsP3 is the messenger, or one of the messengers, involved in transmitterinduced (pharmacomechanical) Ca release from the sarcoplasmic reticulum, which is the intracellular Ca store identified previously as the source of Ca released by norepinephrine in main pulmonary artery.Activation of smooth muscle by transmitters and drugs involves, at least in part, the release of intracellular Ca (1-3), followed by Ca activation ofthe calmodulin-regulated myosin light chain kinase (4). The release of intracellular Ca does not require influx of extracellular Ca (5-7) and can be triggered by pharmacomechanical coupling, a process independent of changes in surface membrane potential (8). Recent electron probe analytic studies have directly demonstrated that the sarcoplasmic reticulum (SR) is the source of intracellular Ca released by norepinephrine in rabbit main pulmonary artery (MPA) (1), portal vein (7), and, probably, other smooth muscles. However, until very recently, the mechanism through which drugs and transmitters released Ca from the SR remained unknown.The recognition, in nonmuscle cells, that stimulation of phosphatidylinositol turnover, stimulated by cholinergic agents and other secretogogues (9, 10), is associated with the production of a metabolite, inositol 1,4,5-trisphosphate (Ins-P3), that can release Ca from the endoplasmic reticulum (11, 12) indicated that InsP3 may also function as an excitatory messenger in smooth muscle. In this study, we demonstrate that InsP3 can, indeed, release Ca from smooth muscle cells of the rabbit MPA, as indicated by Ca2+-selective electrode measurements and by InsP3-induced contraction of vascular strips permeabilized with saponin or digitonin. The quantity of Ca2' released by InsP3, like that released from the SR by norepinephrine in this tissue (1), is sufficient for the activation of contraction. These and other recent studies of phosphatidylinositol turnover (including its stimulation by norepinephrine) and of the effects of InsP3 in smooth muscle (13)(14)(15)(16)(17) provide further evidence for the possibility that InsP3 is a physiological messenger mediating agonist-induced Ca release in smooth muscle. MATERIALS AND METHODSTissue Preparation. Male New Zealand rabbits (1-2 kg of body weight) were killed by a blow on the back of the head...

Magnesium is abundant in the mammalian body and the second most abundant cation in cells. Because the concentration of intracellular free Mg2+ is relatively high (0.2-1 mM), Mg2+ is unlikely to act as a second messenger, like Ca2+, by rapidly changing its cytosolic concentration. But changes in Mg2+ do have profound effects on cellular metabolism, structure and bioenergetics. Key enzymes or metabolic pathways, mitochondrial ion transport, Ca2+ channel activities in the plasma membrane and intracellular organelles, ATP-requiring reactions, and structural properties of cells and nucleic acids are modified by changes in Mg2+ concentration. Yet, although some information is available from giant cells and bacteria, little is known about the regulation of intracellular Mg2+ in mammalian cells. Here we report a new transport mechanism for Mg2+ across the sarcolemma of cardiac cells in both intact hearts and dissociated myocytes. We show that noradrenaline, through beta-adrenergic stimulation and increase of cyclic AMP, stimulates a large efflux of Mg2+ from cardiac cells. This transport is of major dimensions and can move up to 20% of total cellular Mg2+ within a few minutes.

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.

Initial velocities of energy-dependent Ca + + uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca + + uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 pM Ca + + and increased sigmoidally to 8 nmol/mg/s at 200 uM Ca + + . A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca ++ transport in cardiac mitochondria. Comparable rates of Ca+ + uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca + + uptake by frog heart mitochondria were higher at any Ca + + concentrations. The half-maximal rate of Ca + + transport was observed at 30, 60, 72, 87, 92 MM Ca + + for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent Km render mitochondrial Ca + + uptake slow below 10 puM. At these concentrations the rate of Ca ++ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca + + to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca ++ by mitochondria is inadequate for regulating the beat-tobeat Ca + + cycle. The results obtained and the proposed cooperativity for mitochondrial Ca+ + uptake are discussed in terms of physiological regulation of intracellular Ca++ homeostasis in cardiac cells.

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The abundance of magnesium (Mg 2+ ) within mammalian cells is consistent with its relevant role in regulating tissue and cell functions. At the last count, more than three hundred and fifty enzymes, aside from metabolic cycles, appear to require and be regulated by concentrations of Mg 2+ that are well within the physiological range observed in tissues and cells. The absence of detectable major changes in cellular free [Mg 2+ ], and the extremely slow turn-over of the cation across the cell plasma membrane under quiescent condition has supported for more than three decades the assumption that cellular Mg 2+ content is kept constant at the level necessary for enzyme and channel function, and that its concentration does not require drastic and rapid changes to form complex with ATP and other phosphonucleotides.In the last decade, a large body of new experimental observations has significantly reverted this way of thinking.Compelling evidence now suggests that large fluxes of Mg 2+ can cross the cell plasma membrane in either direction following a variety of hormonal and non-hormonal stimuli, resulting in major changes in total and, to a lesser extent, free Mg 2+ content within tissues, and in a marked variation in the opposite direction of circulating Mg 2+ level. The present review will attempt to update our knowledge in this area and provide some insights on how changes in cellular Mg 2+ content can result in a modification of the activity rate for several cellular enzymes. Mg 2+ AS AN INTRACELLULAR MESSENGERFor many decades the role of Mg 2+ in biological systems has been hampered by the difficulty of measuring accurately and selectively Mg 2+ in cell and biological system. This has been partly ameliorated by the introduction of atomic absorbance spectrophotometry in the early 1950s and, more recently, by additional analytical methods. Mg2+ is now recognized being indispensable for enzyme activity and structural modification of phosphometabolites or channels.Yet, the general consensus from a large body of evidence indicates that Mg 2+ concentration is relatively stable within the cell and that whilst Mg 2+ presence is necessary for cell function, it will not modulate -like Ca 2+ -cell function by changing concentrations within the cytsosol.In fact, studies attempting to equate Ca 2+ and Mg 2+ as signaling molecules for cytosolic enzymes have been disappointing. Ca 2+ is a signaling molecule because of the following conditions: a) the free cytosolic concentration is extremely low; b) the concentrations in plasma and cytosolic reservoirs are very high, establishing a large concentration gradient across biological membranes; c) because of the low resting Ca CHANGES IN SERUM MG 2+ LEVELCirculating Mg 2+ level is 1.5-1.7 mEq/L in humans and in many mammals (1-3). A decrease in serum Mg 2+ level has been reported to occur during several chronic diseases, both in humans and in animals (4-6). Yet, there is a remarkable lack of information, or contrasting result, as to whether magnesemia undergoes c...