Brain interstitial fluid calcium concentration during development in the rat: control levels and changes in acute plasma hypercalcaemia

Age-related changes in brain interstitial fluid (ISF) ionic calcium, in ionic and total calcium in plasma and the effect of plasma hypercalcaemia on ISF calcium have been studied in rats aged between late gestation and adult. ISF ionic [Ca2+] decreased significantly with development from 1.6 mM to 1.2 mM. Plasma ionic [Ca2+] was not significantly different from ISF [Ca2+] apart from a transient hypocalcaemia at birth which was not reflected in the ISF. Plasma total calcium was around 2X ionic [Ca2+] and showed the same age-related decrease. In acute plasma hypercalcaemia induced by calcium gluconate injections, there was only weak regulation of ISF Ca2+ at 21 days gestation but a rapid improvement after birth resulted in excellent control by 5 days.     

Free calcium concentrations in Krebs-Ringer bicarbonate buffer: Effects on 45Ca- and 32P-transport across the perfused guinea pig placenta

Calcium ion activity was measured in protein-free and protein-containing Krebs-Ringer bicarbonate buffer. Even in protein-free buffer calcium ion activity was 20% less than the total Ca concentration. In solutions containing albumin the calcium ion activity was dramatically reduced, an effect dependent on the albumin concentration and pH of the solution. Acutely changing calcium ion activity from 0.35 mM to 1.25 mM in a Krebs-Ringer bicarbonate buffer (5.4% albumin) perfusing the fetal side of the guinea pig placenta resulted in an immediate increase in transfer (mother to fetus) of ³²P but no change in ⁴⁵Ca transfer. The ratio of ³²P to ⁴⁵Ca clearance was positively related also to maternal plasma calcium ion activity.     

Fetal insulin and placental 3-O-methyl glucose clearance in near-term sheep

It is difficult, if not impossible, to measure the placental transfer of glucose directly because of placental glucose consumption and the low A-V glucose difference across the sheep placenta. We have approached the problem of quantifying placental hexose transfer by using a nonmetabolized glucose analogue (3-O-methyl glucose) which shares the glucose transport system. We have measured the clearance by using a multisample technique permitting least squares linear computing to avoid the errors implicit in the Fick principle. The placental clearance of 3-O-methyl glucose was measured in the control condition and after the administration of insulin to the fetal circulation. A glucose clamp technique was used to maintain constant transplacental glucose concentrations throughout the duration of the experiment. A control series was performed in which the only intervention was the infusion of normal saline. In these experiments the maternal and fetal glucose concentrations remained constant as did the volume of distribution of 3-O-methyl glucose in the fetus. The maternal insulin concentration remained constant and fetal insulin concentration changed from 11 +/- 2 microU/ml to 355 +/- 51 microU/ml (P less than 0.01). In the face of this large increase in fetal plasma insulin, there was no change in the placental clearance of 3-O-methyl glucose. In the control condition the clearance was 14.1 +/- 1.0 ml/min per kg and this was 13.8 +/- 1.0 ml/min per kg in the high insulin condition. Fetal insulin may change placental glucose flux by decreasing fetal plasma glucose concentrations but does not do so by changing the activity of the glucose transport system.     

Evidence of saturable uptake mechanisms at maternal and fetal sides of the perfused human placenta by rapid paired-tracer dilution: studies with calcium and choline

Rapid uptake and efflux of 45Ca2+ and [3H]choline at the maternal and fetal interfaces of the syncytiotrophoblast in the dually-perfused human placenta was investigated by application of the single circulation paired-tracer dilution method (Yudilevich, Eaton, Short & Leichtweiss 1979). Cotyledons were perfused with Krebs-bicarbonate containing dextran (30 g/l; MW = 60-70,000) at 20 and 6 ml/min on maternal and fetal sides, respectively. The paired-tracer (test substrate and extracellular marker) technique consisted of an intra-arterial injection of a tracer bolus, followed by venous sampling over 5-6 min. There was a rapid (sec) uptake of 45Ca2+, followed by backflux (efflux into the ipsilateral circulation) which, over 5-6 min, was 59-100% on the fetal side. It was more variable but generally lower on the maternal interface. At 0.1 mM calcium, 45Ca2+ maximal uptake (Umax) was about 53% on the fetal side but on the maternal side it was variable and averaged 17%. At 2.4 mM calcium fetal side Umax was reduced to 40%. However, on the maternal side the effect was not consistent. Unidirectional influx (nmol/min per g) appeared to be not different on the two sides of the placenta. For [3H]choline (in choline-free perfusates) Umax was about 50% and 30% on fetal and maternal sides, respectively; tracer backflux was variable on the maternal side and averaged 50% on the fetal side. [3H]Choline uptake was highly inhibited by either 1.0 mM choline or the specific competitive inhibitor, hemicholinium-3 (0.1 mM). Specific transplacental transfer of 45Ca2+ (i.e. in excess of the extracellular marker) was not significant in either direction. For [3H]choline there was an apparent small excess (about 4%) preferential towards the fetal circulation. These findings in the human placenta are similar to those demonstrated previously in the guinea-pig placenta which suggested the existence of specific transport systems for choline and calcium on both sides of the syncytiotrophoblast.     

PHosphate metabolism and foetal growth in the rat. I. Net transfer of 31P and 32P phosphate from the maternal plasma to the normal placenta

Net transfer of 31P and 32P inorganic phosphate form the maternal plasma to the rat chorio-allantoic placenta has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or late stages of gestation. The concentration and label uptake per unit weight of placenta of the inorganic phosphate (Pi), organic-bound acid-soluble phosphate (POAS) and organic-bound acid-insoluble phosphate (POAIS) fractions are negatively correlated with increasing placental weight, whereas their specific activities are independent of placental weight. The amount and label uptake per whole placenta of the Pi, POAS and POAIS fractions are positively correlated with increasing placental weight. The placental concentrations of inorganic phosphate and calcium are positively related without, however, any marked accumulation of calcium. The growing placenta is thus shown to reduce progressively, on a unit weight basis, both the inorganic phosphate uptake from the maternal plasma and its further incorporation into organic-bound fractions. There is no evidence of a control by the foetal weight, acting per se, on these placental activities.     

An in vivo study of ovine placental transport of essential amino acids

Under normal physiological conditions, essential amino acids (EA) are transported from mother to fetus at different rates. The mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta and the regulation of EA concentrations in maternal and fetal plasma. To study the relation of EA transplacental flux to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of concentration and molar enrichment in maternal and fetal plasma and of umbilical plasma flow were used to calculate the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and Val) had relatively high and similar clearances and were followed, in order of decreasing clearance, by Trp, Thr, His, and Lys. The five high-clearance EA showed strong correlation (r(2) = 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have similar affinity for a rate-limiting placental transport system that mediates rapid flux from mother to fetus, and that differences in transport rates within this group of EA are determined primarily by differences in maternal plasma concentration.     

The vitamin D receptor is not required for fetal mineral homeostasis or for the regulation of placental calcium transfer in mice

We utilized a vitamin D receptor (VDR) gene knockout model to study the effects of maternal and fetal absence of VDR on maternal fertility, fetal-placental calcium transfer, and fetal mineral homoeostasis. Vdr null mice were profoundly hypocalcemic, conceived infrequently, and had significantly fewer viable fetuses in utero that were also of lower body weight. Supplementation of a calcium-enriched diet increased the rate of conception in Vdr nulls but did not normalize the number or weight of viable fetuses. Among offspring of heterozygous (Vdr(+/-)) mothers (wild type, Vdr(+/-), and Vdr null fetuses), there was no alteration in serum Ca, P, or Mg, parathyroid hormone, placental (45)Ca transfer, Ca and Mg content of the fetal skeleton, and morphology and gene expression in the fetal growth plates. Vdr null fetuses did have threefold increased 1,25-dihydroxyvitamin D levels accompanied by increased 1alpha-hydroxylase mRNA in kidney but not placenta; a small increase was also noted in placental expression of parathyroid hormone-related protein (PTHrP). Among offspring of Vdr null mothers, Vdr(+/-) and Vdr null fetuses had normal ionized calcium levels and a skeletal ash weight that was appropriate to the lower body weight. Thus our findings indicate that VDR is not required by fetal mice to regulate placental calcium transfer, circulating mineral levels, and skeletal mineralization. Absence of maternal VDR has global effects on fetal growth that were partly dependent on maternal calcium intake, but absence of maternal VDR did not specifically affect fetal mineral homeostasis.     

Transient and Persistent Hypercalcaemia in Patients Treated by Maintenance Haemodialysis

In a group of 32 patients with terminal renal failure the initial hypocalcaemia was corrected after two months'' adequate maintenance haemodialysis. In seven patients hypercalcaemia occurred with a peak incidence after about six months'' treatment. In six of these patients hypercalcaemia was transient and the plasma calcium became normal with haemodialysis alone. In one patient the hypercalcaemia was persistent and the plasma calcium reverted to normal only after subtotal parathyroidectomy. This patient had no radiological bone disease, a normal alkaline phosphatase, and no metastatic calcification of the soft tissues.It is concluded that in some patients with terminal renal failure treated with maintenance haemodialysis autonomy of the parathyroids becomes evident in the absence of bone disease or a raised plasma alkaline phosphatase, and that subsequently with continued dialysis there is a spontaneous involution towards normal parathyroid function.     

Calcium regulation of magnesium dependent phosphorylation of human erythrocyte ghost spectrin

Phosphorylation of human erythrocyte ghost membrane proteins was found to be affected by micromolar calcium concentrations. Increasing Ca2+ concentration to 0.2 microM decreased spectrin (band 2) phosphorylation to 30 +/- 6% of control (to which no calcium was added). Decreasing calcium concentration by adding EGTA (0.2mM) to the standard membrane preparation increased spectrin phosphorylation to 575% control. This effect of Ca2+ was more pronounced at higher temperature. At 0 degree C, Ca2+ (0.05mM) had no effect on protein phosphorylation. Sodium fluoride like EGTA caused a four to five fold increase in phosphorylation. Pyrophosphate, a phosphoprotein phosphatase inhibator, had no effect. Once spectrin was phosphorylated in the presence of [gamma-32P]ATP the addition of Ca2+ or EGTA did not decrease or increase its phosphorylation. It is suggested that calcium regulates spectrin phosphorylation either by decreasing kinase activity or by decreasing substrate availability.     

Ablation of calcitonin/calcitonin gene-related peptide-alpha impairs fetal magnesium but not calcium homeostasis

We used the calcitonin/calcitonin gene-related peptide (CGRP)-alpha gene knockout model (Ct/Cgrp null) to determine whether calcitonin and CGRPalpha are required for normal fetal mineral homeostasis and placental calcium transfer. Heterozygous (Ct/Cgrp(+/-)) and Ct/Cgrp null females were mated to Ct/Cgrp(+/-) males. One or two days before term, blood was collected from mothers and fetuses and analyzed for ionized Ca, Mg, P, parathyroid hormone (PTH), and calcitonin. Amniotic fluid was collected for Ca, Mg, and P. To quantify skeletal mineral content, fetuses were reduced to ash, dissolved in nitric acid, and analyzed by atomic absorption spectroscopy for total Ca and Mg. Placental transfer of (45)Ca at 5 min was assessed. Ct/Cgrp null mothers had significantly fewer viable fetuses in utero compared with Ct/Cgrp(+/-) and wild-type mothers. Fetal serum Ca, P, and PTH did not differ by genotype, but serum Mg was significantly reduced in null fetuses. Placental transfer of (45)Ca at 5 min was normal. The calcium content of the fetal skeleton was normal; however, total Mg content was reduced in Ct/Cgrp null skeletons obtained from Ct/Cgrp null mothers. In summary, maternal absence of calcitonin and CGRPalpha reduced the number of viable fetuses. Fetal absence of calcitonin and CGRPalpha selectively reduced serum and skeletal magnesium content but did not alter ionized calcium, placental calcium transfer, and skeletal calcium content. These findings indicate that calcitonin and CGRPalpha are not needed for normal fetal calcium metabolism but may regulate aspects of fetal Mg metabolism.     

Uptake and release of 45Ca by Myxicola axoplasm

The binding and release of 45Ca by axoplasm isolated from Myxicola giant axons were examined. Two distinct components of binding were observed, one requiring ATP and one not requiring ATP. The ATP- dependent binding was largely prevented by the addition of mitochondrial inhibitors, whereas the ATP-independent component was unaffected by these inhibitors. The ATP-independent binding accounted for roughly two-thirds of the total 45Ca uptake in solutions containing an ionized [Ca2+] = 0.54 microM and was the major focus of this investigation. This fraction of bound 45Ca was released from the axoplasm at a rate that increased with increasing concentrations of Ca2+ in the incubation fluid. The ions Cd2+ and Mn2+ were also able to increase 45Ca efflux from the sample, but Co2+, Ni2+, Mg2+, and Ba2+ had no effect. The concentration-response curves relating the 45Ca efflux rate coefficients to the concentration of Ca2+, Cd2+, and Mn2+ in the bathing solution were S-shaped. The maximum rate of efflux elicited by one of these divalent ions could not be exceeded by adding a saturating concentration of a second ion. Increasing EGTA concentration in the bath medium from 100 to 200 microM did not increase 45Ca efflux; yet increasing the concentration of the EGTA buffer in the uptake medium from 100 to 200 microM and keeping ionized Ca2+ constant caused more 45Ca to be bound by the axoplasm. These results suggest the existence of high-affinity, ATP-independent binding sites for 45Ca in Myxicola axoplasm that compete favorably with 100 microM EGTA. The 45Ca efflux results are interpreted in terms of endogenous sites that interact with Ca2+, Cd2+, or Mn2+.     

Phosphate metabolism and foetal growth in the rat. III. Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the placenta after foetectomy

In primigravid rat females at the 12th day or later stages of gestation one foetus is surgically removed and the amniotic sac allowed to retract freely around the remaining placenta in situ. One hour to 10 days after foetectomy [32P]ortho-phosphate is injected intraperitoneally. The post-foetectomy placenta and matched normal placenta and foetus in the contralateral horn are taken after labelling periods of one or three hours and analysed for calcium, inorganic phosphate, organic-bound acid-soluble and organic-bound acid-insoluble phosphates in their [31P] and [32P] forms. The acutely foetectomized placenta--up to 24 hours after surgery--may serve as an appropriate model of the physiologically functioning syncytiotrophoblast: it is indeed noteworthy that organic-bound phosphate placental concentrations and labelling are little affected in the early post-foetectomy stages. On the other hand the placental inorganic phosphate concentration and labelling are profoundly affected by foetectomy. A surplus radioactive incorporation in the Pi fraction is observed which is strongly related to the weight of the control foetus and may represent up to twelve times the label incorporation in the control placenta; it decreases as the time interval since foetectomy lengthens. A cumulative increase in Pi concentration is observed which is strongly related to calcium concentration and may reach twenty-five times the maternal plasma Pi concentration. The presence of two independent pools of Pi is suggested: one geared to the metabolic needs of the placenta itself, the other representing the Pi normally transported to the foetus under metabolic control by the latter. The possibility of an electroneutral PO4(2-)/Ca2+ co-transport is evoked.     

Studies on alpha-adrenergic activation of hepatic glucose output. Studies on role of calcium in alpha-adrenergic activation of phosphorylase.

The role of Ca2+ ions in alpha-adrenergic activation of hepatic phosphorylase was studied using isolated rat liver parenchymal cells. The activation of glucose release and phosphorylase by the alpha-adrenergic agonist phenylephrine was impaired in cells in which calcium was depleted by ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) treatment and restored by calcium addition, whereas the effects of a glycogenolytically equivalent concentration of glucagon on these processes were unaffected. EGTA treatment also reduced basal glucose release and phosphorylase alpha activity, but did not alter the level of cAMP or the protein kinase activity ratio (-cAMP/+cAMP) or impair viability as determined by trypan blue exclusion, ATP levels, or gluconeogenic rates. The effect of EGTA on basal phosphorylase and glucose output was also rapidly reversed by Ca2+, but not by other ions. Phenylephrine potentiated the ability of low concentrations of calcium to reactivate phosphorylase in EGTA-treated cells. The divalent cation inophore A23187 rapidly increased phosphorylase alpha and glucose output without altering the cAMP level, the protein kinase activity ratio, and the levels of ATP, ADP, or AMP, The effects of the ionophore were abolished in EGTA-treated cells and restored by calcium addition. Phenylephrine rapidly stimulated 45Ca uptake and exchange in hepatocytes, but did not affect the cell content of 45Ca at late time points. A glycogenolytically equivalent concentration of glucagon did not affect these processes, whereas higher concentrations were as effective as phenylephrine. The effect of phenylephrine on 45Ca uptake was blocked by the alpha-adrenergic antagonist phenoxybenzamine, was unaffected by the beta blocker propranolol, and was not mimicked by isoproterenol. The following conclusions are drawn: (a) alpha-adrenergic activation of phosphorylase and glucose release in hepatocytes is more dependent on calcium than is glucagon activation of these processes; (b) variations in liver cell calcium can regulate phosphorylase alpha levels and glycogenolysis; (c) calcium fluxes across the plasma membrane are stimulated more by phenylephrine than by a glycogenolytically equivalent concentration of glucagon. It is proposed that alpha-adrenergic agonists activate phosphorylase by increasing the cytosolic concentration of Ca2+ ions, thus stimulating phosphorylase kinase.     

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.     

III. Prolactin and calcium metabolism in the rat

The relation between plasma prolactin and plasma calcium was investogated in normoprolactinemic and hyperprolactinemic adult male rats. Hyperprolactinemia was induced by implantation of a pituitary underneath the kidney capsule in intact males. Hyperprolactinemia did not affect the concentration of calcium in the plasma. It did neither affect food and water consumption, nor urine production and urinary sodium excretion, indicating that prolactin is not important for osmoregulation. However, in hyperprolactinemic rats urinary calcium excretion was markedly increased. 90 Min of intravenous infusion of CaCl₂ induced a similar rise of plasma calcium in normoprolactinemic and hyperprolactinemic rats. Infusiion of EGTA during 60 min induced no change in total plasma calcium in normo- and hyperprolactinemic rats. although probably ionized plasma calcium was considerably decreased. Plasma prolactin, however, was not changed during these infusions. It is concluded that in the adult male rat prolactin has no function in the short term regulation of calcium homeostasis. However, when plasma prolactin is considerably increased duing prolonged periods, the hormone may affect calcium metabolism.     

Acute cord occlusion increases blood ionized magnesium concentration in preterm fetal sheep during maternal magnesium sulfate exposure

This study tested the hypothesis that a pathophysiologic insult to the fetus that decreases pH (umbilical cord occlusion) produces an increase in physiologically active (i.e., ionized) magnesium concentration. Preterm pregnant sheep (n = 7) were instrumented with maternal and fetal catheters and an inflatable vascular occluder was placed around the umbilical cord. After a 2-day recovery period, each ewe received a 4-g loading dose, followed by continuous intravenous infusion of 1 g magnesium sulfate/h. After 48 h, an episode of acute fetal distress was produced by inflation of the umbilical occluder for 10 min. Maternal and fetal arterial blood samples were collected at regular intervals to quantitate ionized magnesium concentration and monitor physiologic status. Magnesium sulfate infusion increased maternal and fetal blood ionized magnesium concentration. In vitro blood analysis demonstrated that there was a linear inverse correlation (r2 = 0.99) between fetal sheep blood pH and ionized magnesium concentration. In vivo, 10 min of umbilical cord occlusion produced an increase in fetal blood ionized magnesium concentration in all animals (P = 0.02) that was temporally related to the decrease in fetal blood pH. Whether this increase in physiologically active magnesium concentration is beneficial (via neuroprotection) or deleterious (via suppression of stress response) to the distressed fetus remains to be determined.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Concentrations of progesterone in arterial and venous plasma of fetal pigs and their dams in late gestation

Blood samples were drawn from uterine arteries and veins of pregnant gilts and from the umbilical artery and vein of each of their fetuses during laparotomy at Day 80. Concentrations of progesterone (P) were greater in fetal than maternal plasma. Uptake of P from the placenta by the fetal blood was evident but was not equivalent to the maternal uterine arterial-venous difference in P concentration. No correlation between plasma P and fetal weight was noted. Concentrations of P in both umbilical vessels of female fetuses were higher than in male fetuses. These data indicate that fetal sex affects the rate of transport and/or synthesis of P in the utero/placental compartment and/or the rate of metabolism of P in the fetus. The relative importance of de novo synthesis and transplacental transport of P in establishing concentrations of P in fetal blood remains to be elucidated.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.