Adele B. Kostellow

Sequential forms of ATPase activity correlated with changes in cation binding and membrane potential from meiosis to first cleavage in R. pipiens

Abstract Changes in cortical membrane potential, cation binding, subcellular aggregation, ATPase activity, phosphoprotein phosphatase activity and inorganic phosphate levels have been measured in the R. pipiens egg from meiotic prophase to first cleavage. 1. 1. Between meiotic prophase (oocyte) and metaphase of the second maturation division (ovulated egg), the egg cortex depolarizes and the egg cytoplasm becomes positive relative to the external environment. Fertilization is accompanied by a transient positive hyperpolarization and the potential returns to the level of the ovulated egg by the completion of meiosis (15 to 20 min). The egg cytoplasm remains positive until first cleavage when the cortex slowly repolarizes and the cytoplasm again becomes negative relative to the external medium. 2. 2. During the depolarization phase there is an increase in K + , Ca 2+ and Mg 2+ binding to the cortical pigment granule fraction and an increase in soluble Na + , Ca 2+ and Mg 2+ . Concomitant with these ionic shifts there occurs a massive subcellular disaggregation. During the rising phase of the hyperpolarization there is a release of bound Na + , K + , Ca 2+ , and Mg 2+ and a rapid efflux of 15 to 25% of the Na + , K + , and Mg 2+ and 80% of the Ca 2+ . By first cleavage, the cations remaining in the egg are largely recovered in both the yolk platelet and pigment granule fractions. 3. 3. In meiotic prophase the principal ATPase is Na-K-Mg activated, Ca-inhibited and ouabain sensitive, consistent with the negative potential and the K + selectivity of the cell membrane. By metaphase of the second maturation division, ATPase activity has increased five-fold and is now Ca-activated and ouabain insensitive, consistent with the apparent disappearance of active Na + transport. After fertilization the endogenous ATPase activity slowly declines and the Mg-requirement returns. The enzyme again becomes Ca-inhibited and ouabain sensitive by first cleavage.

Mg2+ modulates membrane lipids in vascular smooth muscle: a link to atherogenesis

Epidemiological studies associate low dietary magnesium intake with an increased incidence of ischemic heart disease and sudden cardiac death. We have used proton‐magnetic resonance (1H‐NMR) techniques and Mg2+‐selective electrodes to monitor changes in lipid extracts of aortic and cerebrovascular smooth muscle as extracellular ionized magnesium ion concentration ([Mg2+]o) is lowered. We have found that, within the pathophysiological range of Mg2+ concentrations, fatty acid chain length and double bond content are progressively reduced as [Mg2+]o is lowered. In contrast, the plasmalogen content is progressively increased. A concomitant decrease in fatty acid chain length and double bonds indicates oxidation of double bonds resulting in truncation of the fatty acids. A decrease in lipid oxidation in the presence of elevated Mg2+ could contribute to the apparent protective role of increased Mg2+ intake on vascular function in humans.

Specific binding of progesterone to the cell surface and its role in the meiotic divisions in Rana oocytes

Progesterone is believed to act at the cell surface to induce the resumption of the meiotic divisions in amphibian oocytes. Analysis of [3H]- and [14C] progesterone uptake and exchange by the plasma-vitelline membrane complex, nucleus and cytoplasm of the isolated Rana oocyte indicates that progesterone uptake by the plasma membrane is saturable, specific and temperature-dependent, and has a slow off-rate. Estradiol (a noninducer) did not compete with progesterone, whereas testosterone (an inducer) blocked progesterone uptake by the membrane complex. Scatchard-type plots indicate an apparent Kd of 5.1.10-7 M over the [progesterone]0 range of 0.01-1.0 microM with maximum binding at about 70 fmol per oocyte. Membrane uptake at higher [progesterone]0 (2-40 microM) indicates apparent cooperative binding, with saturation up to 10 pmol per oocyte. Cytoplasmic uptake was apparently nonspecific and less temperature-dependent than membrane uptake and steroid concentrations (progesterone and pregnanediones) exceeded water solubility by 30-60 min. Nuclear uptake was saturable and specific but uptake was independent of temperature. A comparison of membrane binding and a physiological response (nuclear breakdown) indicated only about 10% of the membrane sites need be filled to initiate a 50% response.

Progesterone-induced down-regulation of an electrogenic Na+, K+-ATPase during the first meiotic division in amphibian oocytes

SummaryProgesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na+, K+-pump, and other electrophysiological studies indicate a decrease in both K+ and Cl− conductances of the oocyte plasma membrane. Measurement of [3H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (Kd-4.2×10−8m), K+-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [3H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na+, K+-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.

Mg2+ modulates membrane sphingolipid and lipid second messenger levels in vascular smooth muscle cells.

In vitro studies with smooth muscle cells from rat aorta and dog cerebral blood vessels indicate that variation in free Mg2+, within the pathophysiological range of Mg2+ concentrations, found in human serum, causes sustained changes in membrane phospholipids and lipid second messengers. Incorporation of [3H]palmitic acid into phosphatidylcholine (PC) and sphingomyelin (SM) was altered within 15–30 min after modifying the extracellular Mg2+ ion level ([Mg2+]o). Decreased Mg2+ produced a fall in both [3H]SM and [3H]PC over the first 2 h. After an 18‐h incubation, the [3H]PC/[3H]SM ratio changed from about 20:1 to about 50:1. Increased [Mg2+]o resulted in a 2‐ to 3‐fold increase in [3H]SM compared to only a small increase in [3H]PC over the same period. There was a reciprocal relationship between [3H]ceramide and [3H]1,2‐DAG levels with highest [3H]ceramide and lowest [3H]‐1,2‐DAG levels seen at lowest [Mg2+]o. The results indicate that a fall in extracellular ionized Mg2+ concentration produces a rapid and sustained decrease in membrane sphingomyelin and a moderate rise in intracellular ceramide. A major effect of lowering [Mg2+]o appears to be a down‐regulation of SM synthase. The increased membrane SM content and a concomitant decrease in cell ceramide, in the presence of elevated [Mg2+]o, may be relevant to the apparent protective role of adequate Mg intake on vascular function in humans.

The role of Ca2+ and cyclic nucleotides in progesterone initiation of the meiotic divisions in amphibian oocytes

Abstract Progesterone appears to be the physiological inducer of the resumption of the meiotic divisions in amphibian oocytes. Within minutes following exposure to progesterone there is a release of Ca 2+ and a transient rise in [cAMP] i followed by a fall in [cAMP] i and rise in [cGMP] i over the next 1–3 h. Agents that induce a fall in [cAMP] i induce meiosis whereas those that prevent the fall and/or elevate [cAMP] i block meiosis. A comparison of the conversion of injected [ 3 H]-ATP to [ 3 H]-cAMP and rate of hydrolysis of injected [ 3 H]-cAMP following exposure to meiotic agonists and antagonists indicates that adenylate cyclase and not phosphodiesterase is the rate-limiting step in regulating [cAMP] i in the oocyte. These findings are consistent with a model in which progesterone initiates the resumption of the meiotic divisions by down-regulation of membrane adenylate cyclase via Ca 2+ release from specific membrane sites and that a translocation of Ca 2+ produces a coordinate activation of guanylate cyclase.

Expression of the nuclear factor-κB and proto-oncogenes c-Fos and c-Jun are induced by low extracellular mg2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke

Proto-oncogene (c-fos, c-jun) and nuclear factor-kappa B (NF-kappaB) expression, as well as DNA synthesis, in aortic and cerebral vascular smooth muscle cells (VSMCs) were upregulated by a decrease in extracellular magnesium ions ([Mg2+]o). Upregulation of these transcriptional factors was inversely proportional to the [Mg2+]o and occurred over the pathophysiologic range of serum Mg2+ found in patients presenting with hypertension, ischemic heart disease, and stroke. Removal of extracellular Ca2+ ([Ca2+]o), use of nifedipine or protein kinase C (PKC) inhibitors prevented the upregulation of the proto-oncogenes and DNA synthesis in VSMCs. These data show that [Mg2+]o may be an important, heretofore, overlooked natural modulator of proto-oncogene and NF-kappaB expression in VSMCs and that Ca2+ and PKC may play critical roles in induction of c-fos and c-jun in VSMCs induced by a decrease in [Mg2+]o. These results point to a role for low serum Mg2+ in potential development of hypertension, atherogenesis, vascular disease, and stroke.

Progesterone induces meiotic division in the amphibian oocyte by releasing lipid second messengers from the plasma membrane.

Meiosis in the amphibian oocyte is normally initiated by gonadotropins, which stimulate follicle cells to secret progesterone. The progesterone-induced G2/M transition in the amphibian oocyte was the first well-defined example of a steroid effect at the plasma membrane, since it could be shown that exogenous, but not injected, progesterone induced meiosis and that many of the progesterone-induced changes associated with meiosis occurred in enucleated oocytes. We find that [3H]progesterone binding to isolated plasma membranes of Rana pipiens oocytes is saturable, specific and temperature-dependent. Photoaffinity labeling with the synthetic progestin [3H]R5020 followed by gel electrophoresis demonstrated progestin binding to both 80 and 110 kDa proteins in the oocyte cytosol, whereas only the 110 kDa R5020 binding protein was present in the oocyte plasma membrane. We have shown that progesterone acts at Rana oocyte plasma membrane receptors within seconds to release a cascade of lipid messengers. Membrane-receptor binding causes the successive activation of: 1) N-methyltransferases, which convert phosphatidylethanolamine to phosphatidylcholine (PC); 2) an exchange reaction between PC and ceramide to form sphingomyelin (SM) and 1,2-diacylglycerol (DAG); 3) phospholipase D/phosphatidate phosphohydrolase, releasing a second DAG transient; and 4) phosphatidylinositol-specific phospholipase C, generating inositol trisphosphate and a third DAG transient. Within minutes, diglyceride kinase converts newly formed DAG species to phosphatidic acid, turning off the successive DAG signals. A transient fall (0-30 s) in intracellular ceramide is followed (within 1-2 min) by a sustained rise in intracellular ceramide lasting 3-4 h. This ceramide may be significant in later cyclin-dependent steps. We conclude that the initial action of progesterone at its plasma membrane receptor triggers a series of enzyme activations that modify the membrane and release multiple DAG species.

Role of calcium in regulating intracellular pH following the stepwise release of the metabolic blocks at first-meiotic prophase and second-meiotic metaphase in amphibian oocytes.

31P-NMR has been used to monitor changes in intracellular pH following the sequential release of the block at first-meiotic prophase by hormones and the block at second-meiotic metaphase by fertilization in Rana eggs and oocytes. The broad phosphoprotein signal was eliminated by a combination of spin-echo and deconvolution techniques. pHi was determined from the pH-dependent separation of intracellular Pi and phosphocreatine resonances. Agents that release the prophase block (progesterone, insulin, D-600, La3+) increased pHi from 7.38 to 7.7-7.8 within 1-3 h. Noninducers such as 17 beta-estradiol were without effect. By second-metaphase arrest (ovulated, unfertilized) the pHi had fallen to 7.1-7.2. pHi underwent a transient increase to about 7.7 within the first 30 min at fertilization, with a slow 0.1-0.2 pH unit oscillation during early cleavage. The progesterone-induced elevation of intracellular pH is not blocked by amiloride and occurs in Na+-free medium. A transient rise in pHi occurs when the prophase-arrested oocyte is transferred to Ca2+-free medium or when ionophore A23187 is added to the Ca2+-containing medium. Agents that inhibit the resumption of the first meiotic division either block the rise in pHi (procaine, PMSF) or shorten the time-course of the rise in pHi (ionophore A23187). Conditions that elevate intracellular Ca2+ levels and/or increase Ca2+ exchange produce an increase in pHi, whereas those conditions that decrease intracellular Ca2+ levels and/or exchange produce a fall in pHi within 1 h. The time-course of the increase in pHi both following release of the prophase block and at fertilization coincide with a fall in intracellular cAMP and release of surface and/or intracellular Ca2+. These results suggest that: (1) pHi is a function of cytosolic free Ca2+ levels and/or Ca2+ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca2+ with the resulting changes in pHi and cAMP and resumption of the meiotic divisions.

Kinetics of calcium efflux and exchange from Rana pipiens oocytes immediately following reinitiation of the first meiotic division: Comparison of various meiotic agonists and antagonists

Abstract The reinitiation of the meiotic divisions and the release of 45Ca from the Rana pipiens oocyte has been studied as a function of meiotic agonists and antagonists. Each of the meiotic agonists tested (progesterone, insulin, D-600, La3+) caused a decreased 45Ca uptake and an increased efflux during the first 15 min after exposure. The effects of progesterone, D-600, and La3+ are not additive and progesterone will not release additional 45Ca in oocytes pretreated with D-600 or La3+. Tetracaine inhibits both progesterone-induced release of 45Ca and an early step in meiosis (nuclear membrane breakdown). [Tetracaine]o required for 50% inhibition of nuclear breakdown decreases with decreasing [progesterone]o suggesting competitive inhibition. The Ca, Mg-ionophore A23187 shows a similar competitive inhibition of progesterone-induced nuclear breakdown and stimulates a rapid release of 45Ca within the first 1–3 minutes after exposure to the ionophore. Unlike progesterone, insulin, D-600, or La3+, the ionophore A23187 stimulates both uptake and efflux of 45Ca by oocytes. These results suggest that both a reduced influx and a selective release of calcium from specific membrane sites is essential for steroid reinitiation of the meiotic divisions in R. pipiens oocytes.