Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats.

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of Mg · ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of miltochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg · ATP and 20μM calcium approximately 38 nmol of calcium per mg of microsormal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min. with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 μM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction.Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced calcium uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.Rats were treated with the steroid deoxycorticosterone acetate for ten and thirty days to induce hypertension. After ten days of deoxycorticosterone acetate although hypertension is present, there is no change in calcium uptake activity of aorta microsomes, renal microsomes or renal plasma membranes. After 30 days of deoxycorticosterone acetate treatment calcium uptake activity of renal microsomes is reduced. A variable decrease in calcium uptake activity is observed with aorta microsomes. Renal plasma membrane calcium uptake remains unchanged.     

Regulation of intracellular calcium in chick embryo fibroblast: calcium uptake by the microsomal fraction.

The total membrane fraction of a chick embryo fibroblast (CEF) homogenate accumulates calcium in an energy-dependent manner. This activity can be dissociated into azide-sensitive and azide-insensitive components. The azide-sensitive component of calcium uptake is believed to represent mitochondrial calcium uptake. The azide-insensitive component of calcium uptake is enhanced by the presence of a calcium trapping agent such as oxalate, and cannot utilize, ADP, inorganic phosphate and a Krebs cycle substrate to support uptake. The distribution of the azide-insensitive calcium uptake in subcellular fractions suggests that this uptake occurs in other than mitochondrial membranes. The membranes most likely to contribute to the azide-insensitive component of calcium uptake are the endoplasmic reticulum and plasma membrane. A microsomal preparation from CEF cells is essentially devoid of the azide-sensitive calcium uptake activity. This microsomal activity is similar in characteristics to the sarcoplasmic reticulum of skeletal muscle. However the specific activity of CEF microsomal calcium uptake system is much less than that found in the skeletal muscle system. The transport of calcium by these membranes provide a mechanism for the regulation of cytosol calcium levels and may play a role in the control of movement and growth of cultured cells.     

Regulation of basic fibroblast growth factor binding and activity by cell density and heparan sulfate

The role of cell density in modulating basic fibroblast growth factor binding and activity was investigated. A primary corneal stromal fibroblast cell culture system was used, since these cells do not constitutively express heparan sulfate proteoglycans in vivo except after injury. A 3-5-fold reduction in bFGF binding per cell was observed as cell density increased from 1000 to 35,000 cells/cm2. The cell density-dependent change in bFGF binding was not the result of altered FGFR expression as determined by equilibrium binding experiments and by immunoblot analysis. However, bFGF-cell surface receptor binding affinities were measured to be 10-20-fold higher at low cell densities than at intermediate and high cell density. bFGF-induced cell proliferation was also cell density-dependent, with maximal stimulation of proliferation 190-280% greater at intermediate densities (15,000 cells/cm2) than at other cell densities. This effect was specific to bFGF as serum, epidermal growth factor, and transforming growth factor-beta did not exhibit the same density-dependent profile. Further, heparan sulfate proteoglycans and, specifically, syndecan-4 were implicated as the modulator of bFGF binding and activity. Pretreatment of cell cultures with heparinase resulted in reduced bFGF binding to the cells and abrogated bFGF induced proliferation. These data suggest a mechanism by which cell density regulates heparan sulfate proteoglycan expression and modulates the cellular response to bFGF. Modulation of heparan sulfate proteoglycan expression might be an important aspect of the regulation of stromal cell migration and proliferation during wound healing.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Energy-dependent uptake of ochratoxin A by mitochondria.

The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus, with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [¹⁴C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K_m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m-chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca²⁺ plus P_istimulated respiration and [³²P]P_i incorporation. In turn, phosphate inhibits nearly completely [¹⁴C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P_i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.     

Energy-dependent uptake of arsenite by rat liver mitochondria.

Uptake of arsenite by rat liver mitochondria is energy-dependent, as shown by comparing values without and with either uncoupling agent or respiratory inhibitor present. The uptake is inhibited by mersalyl and N-ethylmaleimide, which can be used as 'stopping' agents to obtain uptake kinetics. At 20 degrees C the process is nearly complete in 1 min. The relation between the quantity in the energized mitochondria and the applied concentration corresponds to at least two different modes of binding of the arsenite. Competition occurs between arsenite and other anions (for example, phosphate) for intramitochondrial accumulation.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Parathyroid hormone induced stimulation of calcium uptake by renal microsomes

Cortical and papillary microsomes prepared from feline kidneys perfused with parathyroid hormone (PTH) showed an enhanced ability to accumulate calcium (Ca⁺²). PTH was unable to stimulate Ca⁺² uptake into microsomes prepared from outer medulla. These data suggest that renal microsomes may be a valid model system for studying the action of PTH on Ca⁺² transport in the kidney.     

Prolactin receptors in cultured rat mammary tumor cells. Energy-dependent uptake and degradation of hormone receptors

After energy depletion by uncouplers of oxidative phosphorylation or inhibitors of electron transport, primary cultures of carcinogen-induced rat mammary tumors have a 2- to 20-fold increase in the number of cell surface prolactin receptors. When energy-depleted cells were treated with 0.15 M NaCl plus 50 mM glycine pH 3, for 1 min at 4 degrees C, 75% of the specific surface-bound 125I-labeled ovine prolactin was removed, but prolactin and its receptor were not destroyed. Using this technique, we found that receptor-bound prolactin can be internalized (becomes resistant to pH 3.0 treatment) and then degraded. The internalization of occupied receptors required energy, was completed 30-60 min before degradation, and was independent of protein synthesis. Hormone degradation (t1/2, 42 min) but not uptake was prevented by NH4Cl or lysosomotropic amines. In the presence of cycloheximide, receptors were lost (t1/2, 62 min) unless such loss was prevented by KCN. After unoccupied receptors were activated by energy depletion, surface receptors were lost when inhibitor was removed and glucose was added. Thus, both occupied and unoccupied prolactin receptors are constantly removed from the cell surface via an energy-dependent uptake mechanism. If the receptor levels are first increased by energy depletion (with or without bound ligand) or if protein synthesis is inhibited, there is a net loss of surface binding sites. Since the receptors reappeared with 15 h after cycloheximide removal, some of the receptors probably are recycled under normal steady state conditions.     

Regulation of 24-hydroxylase activity in mouse skin fibroblast by cholecalciferol derivatives, triamcinolone acetonide and a calcium modulating agent, nicardipine

Mouse skin fibroblasts in culture were used to study the regulation of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) induced 24 hydroxylase (24-OH-ase) under the influence of 3 agents: (1) 24,25-Dihydroxycholecalciferol (24,25(OH)2D3), 62.5 10(-9) M, which led to a significant decrease in the 1,25(OH)2D3-induced 24-OH-ase, probably acted through a nuclear effect mediated by the 1,25(OH)2D3 receptor protein. (2) Triamcinolone acetonide (10(-6)M) which was found to increase the 24-OH-ase enhancement induced by 1.25 and 6.25 nM 1,25(OH)2D3 whereas it did not alter the effect of 31.2 nM 1,25(OH)2D3. (3) A factor which is likely to induce changes in the cell calcium transport or in the Ca pool sizes, i.e. a calcium channel blocker, nicardipine. The effect of 1.25 nM 1,25(OH)2D3 on 24-OH-ase activity was increased by nicardipine (20 microM) which was found to reduce the effect of 6.25 nM 1,25(OH)2D3. The rate of DNA synthesis (measured by [3H]thymidine incorporation) was increased after incubation of fibroblasts with 1,25(OH)2D3 (1.25 nM) plus triamcinolone acetonide (10(-6) M), although it was reduced by nicardipine in comparison with 1,25(OH)2D3 alone. So the effects of these agents on the 1,25(OH)2D3 induced 24-hydroxylase were shown to be independent of the rate of DNA synthesis.     

Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons

The influence of ACTH and some of its N-terminal related peptides was investigated on the uptake of (3H)-2-deoxy-D-glucose in pure cultures of neurons from chick embryo cerebral hemispheres. ACTH influences deoxyglucose uptake in a time and dose-dependent fashion. The stimulation of deoxyglucose uptake is observed after a delay of 6-8 h and requires active protein synthesis. ACTH does not affect deoxyglucose in non-neuronal cells (astroglial cells, hepatocytes, myoblasts, fibroblasts). The effect of various peptide hormones, neuropeptides and growth factors, active in the central nervous system or other tissues, has also been examined. None of these were able to stimulate deoxyglucose uptake, suggesting that the regulation of hexose uptake in neurons is specific for the ACTH-related peptides.     

Correlation of phenylalanine hydroxylase activity with cell density in cultured hepatoma cells.

We have found that the specific activity of phenylalanine hydroxylase varies over at least a forty-fold range during the growth cycle of Reuber hepatoma (H4) cells growing in monolayer culture. The variation has three phases: (1) a very rapid drop in specific activity upon subculturing to a low cell density; (2) a region of low specific activity and (3) after confluency, a rise to a high specific activity. All the results indicate that the cell density in the culture dish is primarily responsible for this fall and rise in activity. Neither conditioning of the growth medium, the rate of cell division, nor enzyme leakage from the cells appear to play a major role in the changes observed. Lactic dehydrogenase specific activity was determined in all experiments; a much smaller, but still cell density-dependent variation was observed for this enzyme.     

Regulation of foreign DNA uptake by mouse spermatozoa

We have studied some features of DNA uptake in both mature and immature mammalian spermatozoa. Mature sperm collected from the cauda epididymis are able to incorporate foreign DNA in a buffer containing only salts and calcium. Immature spermatozoa, however, are unable to bind DNA. This seems to be caused by the lack of a functional receptor in the sperm membrane since once this membrane is disrupted by sonication, DNA can be detected in the postacrosome region of the sperm nucleus, matching the distribution of the mature spermatozoa. Comparison between the DNA binding proteins of mature and immature spermatozoa allowed us to identify two bands that could be part of the putative membrane receptor for the DNA. On the other hand, DNA uptake in mature sperm is prevented by the seminal plasma. We have identified two components of the seminal plasma, a calcium-dependent DNase present in the seminal vesicle fluid and several DNA binding proteins secreted by the ventral prostate, that could account for the inhibitory activity. Taken as a whole, our results indicate that DNA uptake by the mammalian spermatozoa is a very specific and highly regulated phenomenon.     

Regulation of the ATP dependent calcium uptake activity of heart sarcolemmal vesicles by endogenous cytosolic proteins

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by sarcoplasmic reticulum; further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53-66, 1983). The present study examined the effects of the endogenous cytosolic Ca2+ transport inhibitor and its antagonist on ATP-dependent Ca2+ uptake by sarcolemmal vesicles isolated from rat and canine heart. The cytosolic inhibitor caused strong inhibition (up to 97%) of Ca2+ uptake by sarcolemma (SL); this inhibition could be reversed by the cytosolic inhibitor antagonist. Studies on the characteristics of inhibition revealed the following: a) Inhibition was dependent on the concentration of the inhibitor (50% inhibition with approximately 80 micrograms inhibitor protein). b) The inhibitor reduced the velocity of Ca2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca2+ but caused greater than 2-fold decrease in its apparent affinity for ATP. c) The rates of unidirectional passive Ca2+ release from actively Ca2+ loaded SL vesicles were not altered by low concentrations of the inhibitor (less than 100 micrograms/ml) which were effective in producing marked inhibition of Ca2+ uptake; at higher concentrations (greater than 100 micrograms/ml), the inhibitor caused increase in the rates of passive Ca2+ release. These findings demonstrate that the activity of the ATP-driven Ca2+ pump of cardiac SL can be regulated in vitro by endogenous cytosolic proteins.     

Regulation of the Thiobacillus intermedius glucose uptake system by thiosulfate.

Cells of the mixotrophic chemolithotroph (facultative autotroph) Thiobacillus intermedius which have been grown on a glucose-yeast extract medium, a condition in which glucose is used as a source of energy, accumulate the non-metabolizable analogue 2-deoxy-d-glucose against a concentration gradient in a predominantly unchanged state. On the other hand, cells grown mixotrophically on a thiosulfate-glucose medium, a condition in which glucose provides cell carbon but is not used extensively for energy, and in which enzymes of the Entner-Doudoroff pathway are repressed, do not accumulate 2-deoxy-d-glucose significantly. Similarly, cells grown chemolithotrophically on thiosulfate-carbonate do not take up this sugar. Transfer of thiosulfate-yeast extract-grown cells, which lack the capacity to accumulate 2-deoxy-d-glucose, to a glucose-yeast extract medium results in the induction of the concentrative sugar uptake system. The capacity of induced cells to take up 2-deoxy-d-glucose is inhibited by thiosulfate. Thus, the transport system for glucose appears to be regulated in this organism so that the sugar is accumulated only under conditions where it is utilized as a source of energy, and the presence of the preferred energy source leads to both repression and inhibition of the uptake system.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.