Extracellular calcium regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line

The regulation of the cellular distribution of proteoglycans in a clonal rat parathyroid cell line by extracellular Ca2+ concentrations ([Ca2+]e) was studied. Proteoglycans synthesized by the cells metabolically labeled with [35S]sulfate have been shown to be almost exclusively heparan sulfate (HS) proteoglycans (Yanagishita, M., Brandi, M.L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720), which are generally associated with the plasma membrane. The proportion of HS proteoglycans on the cell surface was approximately 20% in 2.1 mM (high) [Ca2+]e, whereas it increased to 50-60% in 0.05 mM (low) [Ca2+]e. Cell-associated HS proteoglycans redistribute in response to changing [Ca2+]e with a t 1/2 less than 4 min; HS proteoglycans appear on the cell surface as [Ca2+]e decreases and disappear from the cell surface as [Ca2+]e increases. Further, HS proteoglycans on the cell surface recycle to and from an intracellular compartment approximately 10 times before their degradation in low [Ca2+]e but do not recycle in high [Ca2+]e. The distribution of newly synthesized HS proteoglycans is regulated by [Ca2+]e but is independent of [Ca2+]e during biosynthesis. In low [Ca2+]e, at least 50% of the HS proteoglycans pulse-labeled for 10 min are transported from the Golgi complex to the cell surface or to the recycling compartment with a t 1/2 of approximately 20 min. Another approximately 10% appear on the cell surface in either low or high [Ca2+]e in a compartment with a long half-life. Addition of Mg2+ or Ba2+ to the low [Ca2+]e cultures had little effect on the distribution of HS proteoglycans. These observations suggest that [Ca2+]e specifically regulates the distribution and recycling of cell-associated HS proteoglycans in the parathyroid cells.     

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro

The minimum and maximum extracellular Ca2+ concentrations required to promote capacitation, the acrosome reaction, hyperactivated motility, zona penetration and gamete fusion in the mouse have been established. The traces of free calcium in Ca2+-deficient medium were shown not to enhance capacitation since the inclusion of EGTA to chelate free ions during a 120 min preincubation failed to alter the kinetics of capacitation from those observed in the absence of EGTA; 1 h after addition of 1.80 mM-Ca2+, both suspensions were highly fertile. Complete capacitation, when suspensions were immediately functional upon the addition of 1.80 mM-Ca2+, required the presence of greater than or equal to 90 microM-Ca2. Considerably higher concentrations were required to initiate optimal sperm responses: acrosome reaction, 900 microM; gamete fusion, 900 microM; hyperactivated motility, 1.80 mM; zona penetration, 1.80 mM. None of these changes was effected when Ca2+ was less than 450 microM. The responses to elevated Ca2+ were dependent on the length of incubation, being initially positive and then negative. A short (30 min) exposure to 3.40 mM-Ca2+ (x 2 the standard) accelerated capacitation, as evidenced by significantly increased acrosome loss, precocious expression of hyperactivated motility and enhanced fertilizing ability when Ca2+ was reduced to 1.80 mM. However, extended (120 min) preincubation irreversibly damaged sperm function. In the presence of 7.20 mM-Ca2+ (x 4), fertilizing ability was inhibited at both 30 and 120 min, despite a high incidence of acrosome loss. The primary deleterious effect appeared to be on motility which was judged to be more erratic than in 1.80 mM-Ca2+, possibly due to elevated intracellular Ca2+. Because of the considerable difference in threshold Ca2+ concentrations, it is now possible to dissociate the Ca2+-dependent events of capacitation from those of the acrosome reaction and motility changes.     

Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes

The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.     

Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line.

We examined recycling of heparan sulfate (HS) proteoglycans and transferrin receptor (Tf-R) in a rat parathyroid cell line. While extracellular Ca2+ concentration ([Ca2+]e) regulates the recycling of HS proteoglycans in parathyroid cells, such that HS proteoglycans only recycle when [Ca2+]e is lowered below physiological levels, recycling of Tf-R occurs equally well both in 0.05 mM (low) and 2 mM (high) [Ca2+]e. Inhibiting endocytosis chemically with phenylarsine oxide or at low temperature (4 degrees C) did not abolish the effects of changing [Ca2+]e on HS proteoglycans in the recycling compartment even though transport of HS proteoglycans from the Golgi complex to the cell surface was inhibited in low [Ca2+]e. Microtubules are not involved in the recycling of HS proteoglycans or of Tf-R since nocodazole did not affect these processes. Inhibiting the increase of intracellular Ca2+ by an intracellular Ca2+ chelator sustained recycling of HS proteoglycans even in the presence of high [Ca2+]e. These observations show that the exocytosis pathway of HS proteoglycans in the recycling compartment is specifically regulated by [Ca2+]e, whereas that for constitutive secretion is not. Therefore, the recycling of HS proteoglycans may be directly related to some functions of parathyroid cells regulated by [Ca2+]e. Although the mechanism by which [Ca2+]e regulates the exocytosis and recycling of HS proteoglycans is uncertain, it is suggested that an increase of intracellular Ca2+ is necessary, but not necessarily sufficient, for inhibiting their exocytosis.     

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.     

Effects of MgCl2 on the release and recycling of heparan sulfate proteoglycans in a rat parathyroid cell line.

Divalent cations, such as Mg2+, Ba2+, and Co2+, are known to mimic the effects of Ca2+ in parathyroid cells, but it is not clear whether the mechanism of their action is the same as that of Ca2+. We have shown that extracellular Ca2+ concentration ([Ca2+]e) regulates the distribution and recycling of cell-surface heparan sulfate (HS) proteoglycans in a rat parathyroid cell line; at normal to high [Ca2+]e (e.g., 2 mM) HS proteoglycans are primarily localized intracellularly, while at low [Ca2+]e (0.05 mM) they are translocated to the cell surface and rapidly recycle (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C., 1990, J. Biol. Chem. 265, 13661-13668). We now show that a high concentration of Mg2+ (8 mM) reduces the amount of recycling HS proteoglycans in low [Ca2+]e. However, the primary effects of high Ca2+ and high Mg2+ on the recycling HS proteoglycans are different. High [Ca2+]e causes translocation of HS proteoglycans to intracellular compartments, while high Mg2+ stimulates cleavage of their core proteins and subsequent shedding of HS proteoglycans into the medium, thereby depleting the recycling molecules. However, high Mg2+ does not induce shedding of HS proteoglycans in high [Ca2+]e. The effects of Ba2+ and Co2+ were similar to those of Mg2+, but Sr2+ showed no significant effects on HS proteoglycan translocation. Otherwise, 8 mM Mg2+ did not alter biosynthesis or intracellular catabolism of HS proteoglycans. These observations suggest that the recycling of HS proteoglycans in parathyroid cells is sensitive only to [Ca2+]e, whereas several other divalent cations can deplete the recycling HS proteoglycans by a distinctly different mechanism. Thus, the mechanism by which Ca2+ regulates the amounts of the recycling HS proteoglycans may be more physiological and play a functional role in parathyroid cells.     

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+.     

Calcium compartmentation and exchange rates in primary hepatocyte culture

We utilized a technique, previously used to study myocardial cells (G. A. Langer, J. S. Frank, and L. M. Nudd, 1979, Amer. J. Physiol. 237, H239-H246), to study 45Ca2+ isotope exchange kinetics in hepatocyte monolayers, cultured on scintillation disks, and perfused in a flow-through chamber. Isolated rat hepatocytes were plated directly on Primaria-coated disks impregnated with scintillation fluors which made up the walls of the perfusion chamber. Following the labeling of the cells with radioactive calcium (45Ca2+), to apparent asymptote, the washout of 45Ca2+ from the cells was measured. A large very fast turnover compartment, as well as small fast and slow turnover compartments, were identified in each experiment. Surface calcium (Ca2+) was determined by its displacement with 1 mM La3+ after asymptote had been reached during 45Ca2+ labeling (1.59 mmol Ca2+/kg dry wt). The rate constant for this compartment was faster than the washout of the chamber (greater than 3.4 min-1 with a t1/2 less than 12 s). The rate constants for the fast and slow exchangeable compartments were 0.11 min-1 (t1/2 = 6.5 min) and 0.013 min-1 (t1/2 = 56 min), respectively. The fast compartment contained 0.40 mmol Ca2+/kg dry wt and the slow compartment contained 0.27 mmol Ca2+/kg dry wt. Neither the fast nor the slow compartment was lanthanum displaceable. Release of 45Ca2+ in response to 100 microM phenylephrine, 10 nM angiotensin II, and 100-microM 2,5-ditert-butyl hydroquinone was measured during the washout phase. Ca2+ released by these compounds was determined to be 0.50 mmol 0.44, and 0.43 mmol Ca2+/kg dry cell wt, respectively. These agents had an effect only during the washout of the fast compartment. In conclusion, this novel technique of on-line measurement of 45Ca2+ exchange in hepatocyte monolayers identified three exchangeable compartments: (1) a very rapidly exchangeable surface compartment, (2) a fast "microsomal" hormone-releasable compartment, and (3) a slow, non-hormone-releasable compartment.     

Oxygen-dependent 1,25-dihydroxycholecalciferol-induced calcium ion transport in rat intestine.

O2-dependent CA2+ uptake by rat duodenal discs has been characterized and used in a revised assay for 1,25-dihydroxycholecalciferol-induced intestinal Ca2+ transport. Although both muscle and mucosal surfaces are exposed in this free-floating-disc assay, the Ca2+ influx across the muscle surface is small, not O2- or vitamin D-dependent, and can be subtracted out. Depriving the animals of food for 9-14 h before assay increases the O2-dependent uptake by about 75%. Half-saturation values for O2-dependent Ca2+ uptake as determined with this assay are: 0.8mM-Ca2+ (fed) and 0.5mM-Ca2+ (food-deprived) for vitamin D-deficient rats, and 0.9mM-Ca2+ (fed) and 1.5mM-Ca2+ (food-deprived) for rats dosed with 1,25-dihydroxycholecalciferol. The maximum velocity of uptake varies from 6.7nmol of Ca2+ per cm2/min (fed) to 7.0nmol of Ca2+ per cm2/min (food-deprived) for vitamin D-deficient rats and 16.7nmol of Ca2+ per cm2/min (fed) to 29 nmol of Ca2+ per cm2/min (food-deprived) for 1,25-dihydroxycholecalciferol-treated rats. By using a 5 min preincubation and 15 min incubation with 1.0mM-Ca2+, duodenal tissue taken from vitamin D-treated rats shows about a 3-fold increase in O2-dependent Ca2+ uptake when compared with tissue taken from vitamin D-deficient animals. The calcium ionophore A23187, depending on concentration, either has no significant effect on or inhibits the O2-dependent uptake, rather than increasing it. Actinomycin D, at a dose of 2 micrograms/g, inhibits the O2-dependent uptake in intestinal discs from both vitamin D-deficient and vitamin D-treated rats by 58 and 80% respectively, when administered in vivo 3 1/2 h before assay.     

500-Kilodalton calcium sensor regulating cytoplasmic Ca2+ in cytotrophoblast cells of human placenta

Two monoclonal IgG antibodies E11 and G11, which react with parathyroid and kidney tubule cells, are in the present communication demonstrated to immunostain the surface of cytotrophoblast cells in human placenta. The G11 but not the E11 antibody has earlier been found to interfere with the sensing and gating of extracellular calcium in parathyroid cells. Microfluorometric measurement of the cytoplasmic calcium (Ca2+i) concentration was performed on suspended placental cells loaded with fura-2. The E11-positive placental cells displayed biphasic and parathyroid-like increases in Ca2+i in response to extracellular Ca2+. This increase was blocked by the G11 antibody and absent in the E11-negative placental cells. A sandwich enzyme-linked immunosorbent assay was constructed in which the G11 and E11 antibodies were shown to react with the same molecule. This calcium sensor was isolated and found to consist of a single, glycosylated polypeptide of approximately 500 kDa.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

Effect of the phorbol ester TPA on PTH secretion. Evidence for a role for protein kinase C in the control of PTH release

Parathyroid hormone (PTH) secretion is stimulated by low extracellular calcium (Ca2+) in association with a reduction in cyosolic Ca2+, indicating that this cell type does not conform to classical models of stimulus-secretion coupling. We used the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate), which directly activates protein kinase C, to investigate the possible role of this enzyme in the unusual secretory properties of the parathyroid cell. TPA causes a dose-dependent stimulation of PTH release inhibited by high extracellular Ca2+ (EC50 = 10 nM) but has relatively little effect on secretion stimulated by low Ca2+. This effect was mimicked by the beta 4-isomer of phorbol 12,13-didecanoate which also activates kinase C, but not by the alpha 4-isomer, which has no effect on this enzyme. TPA does not modify cellular cAMP or cytosolic Ca2+ in the parathyroid cell indicating that its effects on PTH secretion are not mediated indirectly via changes in these second messengers. These results suggest that inhibition of PTH release at high Ca2+ might be related to a reduction in protein kinase C activity which can be overcome when the enzyme is directly activated by TPA.     

Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein

In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells.     

Effect of calcipenia on proteoglycan metabolism and aggregation in normal articular cartilage in vitro.

Glycosaminoglycan synthesis in normal adult dog knee cartilage cultured in medium containing 0, 0.3 MM- and 0.9 mM-Ca2+ was 52, 67 and 78%, respectively, of that in cartilage from the same joints cultured in a normal concentration of Ca2+, i.e. 1.8 mM. Pulse-chase experiments indicated that the rate of degradiation of glycosaminoglycans in cartilage cultured in the absence of Ca2+ was similar to that of glycosaminoglycans in cartilage cultured in 1.8 mM-Ca2+. Although [35S]sulphate incorporation into glycosaminoglycans was decreased in the presence of calcipenia, [3H]leucine incorporation into protein was unaffected. The average hydrodynamic size of newly synthesized proteoglycan aggregates and purified disaggregated proteoglycans from cartilage cultured in the absence of Ca2+ was similar to that of aggregates and disaggregated proteoglycans from cartilage cultured in 1.8 mM-Ca2+.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Evidence that Ca2+ fluxes and respiratory, glycogenolytic and vasoconstrictive effects induced by the action of platelet-activating factor and L-alpha-lysophosphatidylcholine in the perfused rat liver are mediated by products of the cyclo-oxygenase pathway.

The administration of 'acetylglyceryl ether phosphorylcholine' (AGEPC, also known as platelet-activating factor) and L-alpha-lysophosphatidylcholine (LPC) to rat livers perfused with media containing 1.3 mM-Ca2+ was followed by a concentration-dependent efflux of Ca2+ from the liver. Near-maximal response was observed at 100 nM-AGEPC and 50 microM-LPC, and resulted in a net efflux of approx. 130 nmol of Ca2+/g of liver. Onset of Ca2+ efflux occurred about 10 s after AGEPC and LPC administration, reached a maximum after about 50 s (the maximum rate of efflux was approx. 180 nmol/min per g) and thereafter decreased rapidly, and was sometimes followed by a much smaller influx of Ca2+. Sequential infusions of AGEPC or LPC, and phenylephrine, indicate that each of these agents mobilizes Ca2+ from the same intracellular source. The efflux of Ca2+ was not observed in the presence of indomethacin or bromophenacyl bromide, or when the liver was perfused with low-Ca2+-containing (25 microM) media. Other physiological responses, such as changes in respiration, glucose output and portal pressure, were also inhibited under these conditions. The results suggest that the Ca2+-flux changes and other responses are mediated by prostaglandins produced and released within the liver, possibly by cell types other than hepatocytes.     

Suppression of snake-venom cardiotoxin-induced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases.

The incubation of 10(5) single neonatal rat cardiomyocytes with 1 microM-cardiotoxin in a bath medium, Tyrode solution in the presence of 1 mM-Ca2+, at 37 degrees C evoked the following chain of events. Firstly, there appeared a latent period of about 10 min during which the cells behaved normally. Neither lactate dehydrogenase nor ATP leaked from the cells. Cytosolic free Ca2+ increased considerably, as measured by the fluorescence intensity of fura-2-Ca2+ complex. At the same time a large portion of endogenous ATP was depleted. Secondly, after the latent period, the cell beating became irregular and eventually stopped. Thirdly, blebs appeared on the cell surface, leading to cell degeneration. If, before the appearance of blebs, the cells were washed with the bath medium exhaustively or incubated in the presence of the toxin antibody, cytosolic free Ca2+ and endogenous ATP returned to normal levels and cells resumed regular beating. Preincubation of the cells with 3.75 microM-flunarizine or 3.75 microM-diltiazem (both are Ca2+ antagonists), or 1.5 microM-fura-2 acetoxymethyl ester (a chelate for Ca2+), or 200 microM-leupeptin or 50 microM-antipain (both are proteinase inhibitors) considerably suppressed the toxin's ability to degenerate the cells. On the other hand, lysosomal proteinase inhibitor, autophage inhibitor, serine proteinase inhibitor, phospholipase inhibitor and calmodulin antagonist did not inhibit the toxin's activity. The results suggest that the toxin may act on the extracellular surface of intact cardiomyocytes to increase cytosolic free Ca2+. The subsequent cell degeneration may result from the activation of a Ca2+-dependent non-lysosomal proteolytic system.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.