Binding sites for horseradish peroxidase on the cell surface

Summary The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH₃ cells, and isolated rat liver cells was suppressed by 30 M monosialoganglioside, by 30 M trisialoganglioside, or by 5 mM CMP-neurminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyltransferase. The addition of 2 mM Mn²⁺ to the incubation medium with HRP suppressed the reaction for surfacebound HRP, and the addition of 10–20 mM Ca²⁺ intensified the reaction. The addition of 2 mM Zn²⁺ caused less inhibition than that of 2 mM Mn²⁺, and the addition of 2 mM Co²⁺ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells. Suppression of binding by certain nucleotides and glycoproteins, and a role for calcium

Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and post-fixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding reaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and alpha-lactalbumin were strong inhibitors. No addition of Ca2+ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H2O2-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, eLH, and hCG) did not do so. Addition of Ca2+ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calcium, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that alpha-lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Adenylate cyclase in silkworm. Effects of adenosine 3'-phosphate and 2'-deoxyadenosine 3'-phosphate on the enzyme system in fat body

Adenosine 3'-phosphate and 2'-deoxyadenosine 3'-phosphate inhibit silkworm fat body adenylate cyclase. The inhibition has a rapid onset, and is dependent on the concentration of Mn2+ or Mg2+. The concentrations of 2'-deoxy-3'-AMP required for 50% inhibition (Ki) are 13 microM with 2 mM Mn2+ and 32 microM with 10 mM Mg2+. These Ki values are 7-30 times lower than that for 2'-deoxyadenosine. Stimulation of adenylate cyclase by NaF renders the activity more sensitive to the nucleotide inhibition, reducing the Ki value to 4 microM in the presence of Mn2+. The inhibitory activity is specific for adenine 3'-nucleotide; Ki for 2'-AMP and 5'-AMP are ten times or more higher than that for 3'-AMP, and the other 3'-nucleotides including 8-bromo-3'-AMP, 3'-IMP and 3'-GMP have little or no inhibitory activity.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells. Application of a micro-method for the semi-quantitation of surface-bound HRP

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective metal ion binding at the calcium-binding sites of the sea urchin extraembryonic coat protein hyalin

The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 microM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 microM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2(+)-hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2(+)-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration.     

Competition between glycoprotein hormones and horseradish peroxidase for mannose-specific binding sites in cells of endocrine organs

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in paraformaldehyde-fixed, frozen sections of endocrine organs by a cytochemical method reported previously. In the testis, HRP was bound to interstitial cells, probably macrophages, and to sites extending along the surface of spermatozoa in the seminiferous tubules. In the epididymis, cells in the connective tissue, probably fibroblasts or macrophages, showed the specific reaction. In the ovaries, the reaction for lectin-bound HRP was observed in connective tissue cells of the theca externa, and in the mucosa of the uterus, binding of HRP occurred to many fibroblasts. The glycoprotein was also bound to cells in the connective tissue of the thyroid, probably mast cells, as well as to endothelial cells in the adrenal medulla and cortex. In all cases, the binding reaction required Ca2+ and was suppressed by mannose or mannan. Partially purified and highly purified preparations of glycoprotein hormones [ovine follicle-stimulating hormone, ovine luteinizing hormone, bovine thyroid-stimulating hormone, and human chorionic gonadotropin] as well as bovine thyroglobulin and yeast invertase competed with the binding of HRP to all the cells mentioned thus showing that the hormones were bound to the same sites as HRP. When 1 microM HRP was present in the incubation medium, the addition of 15-25 microM of highly purified hormones almost suppressed the reaction for lectin-bound HRP and competitive effects could be observed at even lower concentrations of the hormones.     

Role of vacuolar ion pool in Saccharomyces carlsbergensis: potassium efflux from vacuoles is coupled with manganese or magnesium influx.

Saccharomyces carlsbergensis cells accumulated Mn2+ (or Mg2+) ions in the presence of glucose, fructose, or mannose, but not of deoxyglucose, 3-O-methylglucose, and sorbose. Accumulation of one equivalent of Mn/2+ was coupled with the efflux of two equivalents of K+ from the cells. Mg/2+ did not exit during Mn2+ uptake. Preliminary treatment of cells with various proton conductors or glucose led to the loss of K+ and to the proportional inhibition of Mn2+ uptake. Polyene antibiotic candicidin together with glucose elicited rapid efflux of K+ and completely inhibited Mn2+ accumulation. Exogenous K+ (more than 1 mM), 100 microM N,N'-dicyclohexylcarbodiimide, and 30 mM sodium arsenate inhibited both K+ efflux and Mn2+ influx. K+ efflux from S. carlsbergensis cells affected the vacuolar pool of K+ both during the accumulation of Mn2+ or Mg2+ and during glucose uptake.     

Role of calcium and the calcium-calmodulin complex in resumption of meiosis, cumulus expansion, viability and hyaluronidase sensitivity of bovine cumulus-oocyte complexes

The necessity of calcium (Ca2+) and the Ca2+-calmodulin complex for resumption and completion of meiosis, expansion of cumulus cells, viability and hyaluronidase sensitivity of in vitro cultured bovine cumulus-oocyte complexes was examined by inhibition of the Ca2+-calmodulin complex with eight graduated doses of trifluoperazine (TFP) and by Ca2+ deficiency or depletion. Doses of TFP greater than 2.5 microM decreased the percent of cumulus complexes surviving culture and oocytes completing meiosis, whereas cumulus expansion was unaffected until the cultures contained a near lethal dose (greater than 10 microM). Hyaluronidase caused dispersion of cumulus cells whenever they were expanded regardless of TFP dose. In TC-199 media the completion of meiosis I was suppressed by 0.1 to 1 mM ethylenediaminotetraacetic acid (EDTA) (P less than 0.05) and drastically reduced by 1.0 mM (P less than 0.05). Viability of the cumulus-oocyte complex was not reduced until the dose of EDTA was increased to 1.0 mM (P less than 0.0001). Cumulus expansion was also not suppressed until the dose of EDTA reached 1.0 mM (P less than 0.05). In Ca2+-free (CF) basal media Eagles, completion of meiosis I was reduced by all doses of EDTA (P less than 0.05), whereas viability of the cumulus-oocyte complex was decreased by Ca2+ deficiency or by EDTA addition to basal media Eagles (P less than 0.01). Cumulus expansion was unaffected by Ca2+ removal or chelation. In all experiments, oocytes which were not degenerate underwent germinal vesicle breakdown regardless of treatment.     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells

Summary Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and postfixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6 phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding eaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and -lactalbumin were strong inhibitors. No addition of Ca²⁺ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H₂O₂-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, cLH, and hCG) did not do se. Addition of Ca²⁺ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calciam, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that -lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

Ascorbate protects against tert-butyl hydroperoxide inhibition of erythrocyte membrane Ca2+ + Mg2(+)-ATPase

The incubation of erythrocyte suspensions or isolated membranes containing a residual amount of hemoglobin (0.04% of original cellular hemoglobin) with tert-butyl hydroperoxide (tBHP, 0.5 mM) caused significant inhibition of basal and calmodulin-stimulated Ca2+ + Mg2(+)-ATPase activities and the formation of thiobarbituric acid reactive products measured as malondialdehyde. In contrast, the treatment of white ghosts (membranes not containing hemoglobin) with tBHP (0.5 mM) did not lead to appreciable enzyme inhibition within the first 20 min and did not result in malondialdehyde (MDA) formation. However, the addition of either 10 microM hemin or 100 microM ferrous chloride + 1 mM ADP to white ghosts produced hydroperoxide effects similar to those in pink ghosts (membranes with 0.04% hemoglobin). The concentrations of hemin and ferrous chloride which caused half-maximal inhibition of Ca2+ + Mg2(+)-ATPase activity at 10 min were 0.5 and 30 microM, respectively. The effects of several antioxidants (mannitol, thiourea, hydroxyurea, butylated hydroxytoluene, and ascorbate) were investigated for their protective effects against oxidative changes resulting from tBHP treatment. Over a 30-min incubation period only ascorbate significantly reduced the enzyme inhibition, MDA formation, and protein polymerization. Thiourea and hydroxyurea decreased MDA formation and protein polymerization but failed to protect against the enzyme inhibition. Butylated hydroxytoluene was similar to thiourea and hydroxyurea but with better protection at 10 min. Mannitol, under these conditions, was an ineffective antioxidant for all parameters tested.     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry

The effect of fendiline, a documented inhibitor of L-type Ca2+ channels and calmodulin, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca2+ probe. Fendiline at 5-100 microM significantly increased [Ca2+]i concentration-dependently. The [Ca2+]i rise consisted of an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca2+ influx and internal Ca2+ release. In Ca(2+)-free medium, pretreatment with 50 microM fendiline nearly abolished the [Ca2+]i rise induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca2+. This indicates that the internal Ca2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 microM, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La3+ by 50%, implying that fendiline-induced Ca2+ influx has two components separable by La3+. Consistently, 0.1 mM La3+ pretreatment suppressed fendiline-induced [Ca2+]i rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 50-100 microM fendiline in Ca(2+)-free medium. However, 50-100 microM fendiline inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 inhibited 50 microM fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 microM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca2+]i in MDCK cells by releasing internal Ca2+ in a manner independent of inositol-1,4,5-trisphosphate (IP3), followed by external Ca2+ influx.     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Characterization of the deoxyribonuclease activity of diphtheria toxin

Having discovered that the A domain of diphtheria toxin exhibits intrinsic nuclease activity (Chang, M. P., Baldwin, R. L., Bruce, B., and Wisnieski, B. J. (1989) Science 246, 1165-1168), we proceeded to examine the requirements for optimal enzymic expression. In vitro assays with linear double-stranded DNA demonstrated that optimal activity occurs at pH 7.5 and 37 degrees C. A characterization of the stringent cation-dependence of the reaction revealed increasing activity with increasing Mn2+ up to 30 mM. In contrast, activity levels with Ca2+ or Zn2+ alone peaked at 100 microM and with Mg2+ alone at 1 mM. The Zn2(+)- and Mg2(+)-stimulated activities appear to be dependent on trace amounts of Ca2+. Indeed, inclusion of 2 mM Ca2+ plus 3 mM Mg2+ in the reaction buffer promoted a high level of DNA cleavage even though very little cleavage was seen with either cation alone at 2-3 mM. Addition of 20-200 mM NaCl or KCl caused progressive inhibition. Detection of diphtheria toxin nuclease activity under physiologically relevant conditions suggests that it may be operative in vivo and supports our contention that diphtheria toxin-induced cytolysis is not a simple consequence of protein synthesis inhibition, but rather the final step in a cytolytic pathway linked to chromosomal integrity.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Participation of calcium in flagellar shortening and regeneration in Chlamydomonas reinhardii

Cation chelators cause flagellar shortening in Chlamydomonas reinhardii. Most effective are EDTA and EGTA (1 mM) but pyrophosphate (10 mM) also is effective. Addition of 5 mM Ca2+ after shortening caused by 4 mM EGTA results in flagellar regeneration. Other divalent cations can replace Ca2+ with the following relative activities: Ca2+ greater than Sr2+ = Mn2+ much greater than Ba2+ = Mg2+. Although the specific ion requirement to reverse shortening is not clear, it is possible that all of the ions act by displacing one bound cation, presumably Ca2+. A specific requirement for Ca2+ in flagellar regeneration could be demonstrated, however, because as little as 50 microM EGTA in the presence of 500 microM Mg2+ delayed regeneration and prevented full regeneration. Ca2+ at 100 microM overcame this inhibition.     

Regulatory changes in the K+, Cl- and water contents of HeLa cells incubated in an isosmotic high K(+)-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.     

Effects of calcium antagonists, calmodulin antagonists, and methylated xanthines on polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta

We have studied the ability of fertilized eggs of Ilyanassa obsoleta to undergo polar lobe formation and cytokinesis in the presence of Ca2+ antagonists (Ca2+ channel blockers, Ca2+ uptake inhibitors). Earlier work had suggested little need for exogenous Ca2+ during these cellular shape changes. Again it appears that exogenous Ca2+ probably is not required, based on cell ability to undergo the shape changes with no, or only minor, delay in the presence of 50 mM La3+ at pH 6.5, 10 mM concentrations of Ni2+ or Co2+, 1 mM Cd2+, and 100 microM concentrations of Mn2+, papaverine, verapamil, D600, or diltiazem. In nominally Ca2+-free seawater (containing approximately 10 microM Ca2+) (CFSW), there still is no effect of Cd2+ (up to 100 microM), Ni2+, Co2+, Mn2+, or diltiazem; however, papaverine, verapamil, and D600 in CFSW cause longer delays in the shape changes than they do in the presence of normal levels of Ca2+ (SW). In 10-50 microM nifedipine, shape changes are progressively delayed to the same extent in both SW and CFSW, but more so in CFSW at concentrations above 50 microM nifedipine. Among calmodulin antagonists, trifluoperazine up to 100 microM was without effect, but chlorpromazine at 25-100 microM and calmidazolium at 50-100 microM caused substantial, concentration-dependent delays in the starting times for the shape changes. Methylxanthines caused a substantial speed-up in the starting times for both polar lobe formation and cytokinesis. The most effective of these, caffeine, at optimal concentrations of 0.7-10 mM in SW or CFSW caused shape changes to occur 12-15 min earlier than in controls undergoing a normal 50-min cycle. Caffeine is known to cause release of Ca2+ from muscle sarcoplasmic reticulum. A putative antagonist of intracellular Ca2+ mobilization, TMB-8, significantly inhibited the shape changes of the Ilyanassa cells, whereas a variety of inhibitors of exogenous Ca2+ uptake noted above did not inhibit. We conclude that Ca2+ may be necessary for polar lobe formation and cytokinesis in Ilyanassa cells, but that it may be released from intracellular, sequestered stores rather than derived from exogenous sources.