The first product of phospholipid N-methylation, phosphatidylmonomethylethanolamine, is a lipid mediator for progesterone action at the amphibian oocyte plasma membrane.

Progesterone has been shown to act at plasma membrane receptors on the amphibian oocyte to trigger a cascade of changes in membrane phospholipids and to initiate the G(2)/M transition of the first meiotic division. The earliest event (0-1 min) is the transient N-methylation of phosphatidylethanolamine (PE) to form phosphatidylmonomethylethanolamine (PME), demonstrated using [(3)H]glycerol to prelabel oocyte plasma membrane PE. [(3)H]Glycerol-labeled PME rises 10-fold within the 1-2 min after exposure to progesterone and accounts for conversion of about 50% of the [3H]Glycerol-labeled PE. [(3)H]PME levels slowly decline over the following 10-30 min. [(3)H] or [(14)C] labeled fatty acid experiments showed that newly formed PME is enriched in linoleic or palmitic, but not in arachidonic acid, indicating that specific PE pools undergo progesterone-induced N-methylation. Two plasma membrane changes: activation of serine protease, and Ca(2+) release from the oocyte surface coincide with PME formation; both are prevented by pretreatment of oocytes with the N-methylation inhibitor, 2-methylaminoethane. Media containing PME micelles release both protease and Ca(2+) from intact oocytes within the first 1-2 min. The immediate downstream metabolites of PME, PDE and PC, do not induce serine protease activity or Ca(2+) release. We conclude that progesterone initially activates N-methyltransferase in the oocyte plasma membrane, and that the first product, PME, is responsible for activation of serine protease in the plasma membrane and the release of Ca(2+) from the oocyte surface.

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.

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.

Progesterone release of lipid second messengers at the amphibian oocyte plasma membrane: role of ceramide in initiating the G2/M transition.

Treatment with either sphingomyelinase (SMase), or soluble forms of ceramide, has been reported to induce meiosis in oocytes from Xenopus laevis, a species which can breed throughout most of the year. In this paper the sphingomyelin-derived second messenger, ceramide, is compared with progesterone for its ability to induce meiosis in oocytes from the seasonal breeder, Rana pipiens. Serum gonadotropin levels normally rise as Rana females emerge from hibernation in the spring, stimulating follicular synthesis of progesterone and subsequent ovulation. Injection of gonadotropins can induce earlier meiosis and ovulation, effective from the previous October through the following spring. During the same period, defolliculated oocytes respond to exogenous progesterone by meiosis, as indicated by nuclear breakdown. We find that in the spring, treatment of defolliculated Rana oocytes with exogenous C2- or C8-ceramide or SMase did induce meiosis, but not during the fall or winter. A 50% response was seen by late April and a 100% response by early May. Exposure of [3H]palmitate-labeled Rana oocytes to either exogenous progesterone or to SMase produced a rapid and comparable release of intracellular [3H]ceramide within 1-2 min in fall, winter or spring. Our results from this and from previous experiments indicate that increased ceramide is not the initiating event in meiotic induction in Rana, but is associated with a subsequent pathway which depends upon a threshold level of progesterone.

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.

Progesterone triggers the rapid activation of phospholipase D in the amphibian oocyte plasma membrane when initiating the G2/M transition.

Previous reports indicate that, in the Rana pipiens oocyte, progesterone triggers a rapid rise in 1,2-diacylglycerol (DAG) derived from phosphatidylcholine (PC) in the plasma membranes. This DAG transient, which appears and is terminated within 60-90 s, is derived both from a phospholipase which we assumed to be phospholipase C and from sphingomyelin (SM) synthase. We now find that progesterone stimulates PC and DAG turnover primarily via the phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) pathways as well as via the SM-ceramide pathway. Rana oocytes were prelabeled with [3H]choline chloride under conditions in which about 70% is incorporated into PC of the plasma membrane of the intact oocyte or with [3H]lysoplatelet activating factor (1-O-octadecyl-sn-glycero-3-phosphocholine, lysoPAF) which is selectively incorporated into plasma membrane PC. Progesterone induced the release of [3H]choline from intact oocytes into the medium within 60-90 s. This choline release was dose-dependent and was not inhibited by a putative PC-specific phospholipase C inhibitor, D609. Progesterone also induced a transient rise in [3H]lysoPAF-derived [3H]DAG within 1-2 min followed by a rise in [3H]PA. In the presence of 20 mM ethanol, progesterone stimulated formation of [3H]lysoPAF-derived phosphatidylethanol, indicating progesterone activation of PC-specific PLD and concomitant formation of PA. A DGK inhibitor (D102) reduced the level of [3H]PA, produced a sustained rise in [3H]DAG and was a weak inducer of meiosis in oocytes not exposed to progesterone. A PA phosphohydrolase inhibitor (propranolol) elevated [3H]PA and completely inhibited the progesterone-induced rise in DAG. Progesterone thus acts at oocyte plasma membrane receptors to release PC-derived DAG via both SM synthase and PC-PLD. The duration of the DAG signal is regulated by the coordinate action of DGK and PAP.

Differences in hydration state of nucleus and cytoplasm of the amphibian oocyte.

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 microm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening taue (spin echo time) to 9 msec (from 18 msec) nor lengthening taur (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T1 (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T1 = 3.0 sec), cytoplasm (T1 = 0.8 sec) and nucleoplasm (T1 = 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments.

Progesterone-induced phospholipid N-methylation and sphingomyelin synthesis in the amphibian oocyte plasma membrane: a second source of the 1,2-diacylglycerol second messenger associated with the G2/M transition.

The effects of progesterone and GTP gamma S on phospholipid N-methylation and sphingomyelin synthesis were studied in plasma-vitelline membranes isolated from amphibian (Rana pipiens) oocytes. Plasma-vitelline membranes were preincubated with S-adenosyl-L-[methyl-3H]methionine for 2 min at 20 degrees C and total phospholipids extracted at 0, 15, 30 and 60 s after addition of progesterone and/or GTP gamma S. Progesterone levels (3 microM) that induce meiosis in the intact oocyte stimulated [3H-methyl]incorporation into phosphatidylmonomethylethanolamine (PME) 9-10-fold over the first 60 s, with smaller increases in phosphatidyldimethylethanolamine (PDE) and phosphatidylcholine (PC). [methyl-3H] labeling of sphingomyelin (SM) rises after 30 s, approaching that of [methyl-3H]PME by 60 s. 17 beta-Estradiol, a noninducer of meiosis, was inactive. When oocytes were prelabeled with [3H]palmitic acid, it was found that a fall in [3H]ceramide coincides with the transient increase in [3H]SM, indicating that the end product of N-methylation (PC) undergoes a transfer reaction with ceramide to form SM and 1,2-DG. GTP gamma S levels previously reported to stimulate PC-specific phospholipase C activity in oocyte plasma membranes (5 microM) also stimulated both [methyl-3H]PME and [methyl-3H]SM formation. An inhibitor of phospholipid N-methylation, 2-(methyl-amino)ethanol, blocked stimulation of [methyl-3H]SM synthesis by both progesterone and GTP gamma S as well as induction of meiosis by progesterone. Progesterone thus acts at the oocyte plasma membrane to stimulate PE N-methyltransferase and SM synthase. The finding that GTP gamma S mimics progesterone suggests that N-methyltransferase is mediated by G-protein(s). The transient increase in 1,2-DG which we had previously reported to occur within 1-2 min following progesterone stimulation of the Rana oocyte appears to arise from PC by two different pathways: SM synthesis and hydrolysis of PC by phospholipase C.

Cyclic AMP binding to the amphibian oocyte plasma membrane: possible interrelationship between meiotic arrest and membrane fluidity.

Cyclic AMP, which maintains the vertebrate oocyte in prophase arrest under physiological conditions, exhibits specific and saturable binding to the cytoplasmic face of the prophase-arrested Rana pipiens oocyte plasma membrane. Scatchard type analyses of [3H]cAMP binding to isolated plasma membranes indicate a single class of binding sites with a Kd = 19.3 +/- 7.0 nM at cAMP concentrations below 10(-6) M and additional low affinity site(s) and/or non-specific binding at concentrations above 10(-6) M. Photoaffinity labeling of prophase oocyte plasma membranes with [32P]-8-N3cAMP demonstrates cAMP/cGMP-displacable binding of 8-N3[32P]cAMP to a 100-110 kDa peptide doublet. Plasma membrane fluidity was monitored by electron spin resonance in isolated plasma-vitelline membranes using a 5-doxyl stearic acid probe. Exogenous dibutyryl cAMP (dbcAMP) produces an increase in membrane fluidity within minutes and blocks and/or reverses the progesterone-induced decrease in plasma membrane fluidity. The dbcAMP concentration that produced half-maximal fluidity increase (10 microM) corresponds to the half-maximal inhibiting dose of dbcAMP for progesterone induction of meiosis. Cholera toxin, which elevates intracellular cAMP and blocks meiosis, also increases membrane fluidity and inhibits progesterone-induced decrease in membrane fluidity. Elevated levels of intracellular cAMP thus appear to maintain meiotic arrest by binding to specific plasma membrane site(s) and maintaining the plasma membrane in a relatively fluid state. The progesterone-induced fall in intracellular cAMP first reported in Rana thus appears to be responsible for the progesterone-induced increase in membrane fluidity and further suggests that the change in membrane order is essential for the resumption of the meiotic divisions.

Steroid action at the plasma membrane: progesterone stimulation of phosphatidylcholine-specific phospholipase C following release of the prophase block in amphibian oocytes.

Progesterone, acting at the amphibian oocyte plasma membrane, triggers the progression of the prophase oocyte nucleus through the first meiotic metaphase. We previously reported a transient increase in 1,2-diacylglycerol (1,2-DG) within the first 1-2 min after exposure of Rana pipiens oocytes to progesterone. We have now investigated the source of the 1,2-DG, using this highly synchronous oocyte population. Phospholipid pools of intact prophase-arrested oocytes were labeled with [3H]glycerol, [methyl-3H]choline chloride or 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine (lyso platelet activating factor, lysoPAF). [3H]LysoPAF is selectively taken up into the plasma membrane of the intact oocyte and esterified to form the [3H]alkyl-analogue of phosphatidylcholine (PC). Intact oocytes and/or isolated plasma membranes were then stimulated with progesterone and the changes in [3H]DG, [methyl-3H]phosphocholine and [3H]phospholipids were monitored as a function of time. Progesterone induced a transient increase in [3H]glycerol-derived DG, [methyl-3H]phosphocholine and [3H]alkyl-2-acylglycerol from [3H]alkyl-PC within the first 2 min, indicating activation of a PC-specific phospholipase C. Different pulse-labeling conditions indicate a biphasic rise in [3H]DG from [3H]glycerol-labeled oocytes; the first rise (1-2 min) when phospholipid labeling in the plasma membrane is enriched followed by an approximately 3-fold larger rise at 5-15 min when phospholipids of intracellular membranes are preferentially labeled. An early transient increase in [3H]DG or [3H]alkyl-2-acylglycerol was also seen when progesterone and/or guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) were added to isolated plasma-vitelline membranes prepared from oocytes prelabeled with either [3H]glycerol or [3H]lysoPAF. Progesterone thus appears to activate a G-protein-linked PC-specific phospholipase C in the oocyte plasma membrane which is followed by much larger DG release from intracellular membranes. The transient character of the hydrolysis suggests that this may represent a mechanism for transducing a membrane event into a meiotic signal.

Progesterone-induced second messengers at the onset of meiotic maturation in the amphibian oocyte: interrelationships between phospholipid N-methylation, calcium and diacylglycerol release, and inositol phospholipid turnover.

The steady-state turnover in phospholipid N-methylation, 1,2-diacylglycerol and inositol phospholipids in prophase-arrested Rana pipiens oocytes was compared with changes occurring in these pathways immediately following progesterone induction of the first meiotic division. Oocytes were preincubated with [3H-methyl]methionine, [3H]glycerol, [3H]myo-inositol or [3H]arachidonic acid. Ca2+ efflux was measured in oocytes preloaded with 45Ca2+. Membrane phospholipids and cytosolic levels of radiolabeled 1,2-diacylglycerol (DAG), inositol bis- (InsP2), tris- (InsP3), and tetrakisphosphate (InsP4) were monitored immediately following induction with progesterone. A transient increase in both N-methylation of ethanolamine phospholipids and in [3H]DAG coincides with a release of 45Ca2+ from the oocyte surface during the first minute. At least 80% of the total phospholipid N-methylation is associated with the plasma membrane. 45Ca2+ and [3H]DAG release occur prior to a rise in intracellular InsP3, the latter beginning 2-3 min after exposure to the hormone and reaching a maximum by 15-30 min. Progesterone induces rapid and successive changes in ethanolamine, choline, and inositol-containing phospholipids, which represent three of the four major phospholipid classes found in membranes. The maintenance of higher levels of DAG and InsP3 during the first 90 min might be expected to sustain the previously observed increase in protein kinase C activity.

Progesterone induces transient changes in plasma membrane fluidity of amphibian oocytes during the first meiotic division.

Progesterone acts at the surface of the amphibian oocyte to induce resumption of the meiotic divisions. Progesterone binding leads to a transient dose-dependent decrease in the fluidity (increase in order parameter) of the Rana oocyte plasma membrane, which was detected by electron spin resonance in isolated plasma membranes using either 5- or 16-DOXYL stearic acid probes. The 5-DOXYL probe, which inserts into the membrane with the spin label nearest the surface, showed an increase in the order parameter within minutes, a maximum change by 2 h, and a return to control levels by 6 h. The order parameter for the 16-DOXYL probe, which reflects the fluidity deeper within the plasma membrane, increased slowly and remained elevated during the first meiotic division. RU 38486, a synthetic steroid that blocks progesterone receptors, prevents progesterone-induced fluidity changes. These findings indicate that the binding of progesterone to its receptor changes the oocyte plasma membrane structure resulting in a differential decrease in mobility near the membrane surface compared to that deeper in the membrane.

Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: an early event in initiating the meiotic divisions.

Plasma membranes isolated from Rana oocytes showed a 7-10 fold increase in the Ca2+-dependent phosphorylation of endogenous protein following exposure to meiotic stimuli (progesterone, insulin) either in vivo or in-vitro. Exogenous phosphatidylmonomethylethanolamine (PME) was effective in stimulating Ca2+-dependent membrane phosphorylation and also induced meiosis. Induction of phosphorylation was blocked by the protease inhibitor leupeptin, as are all other responses to meiotic stimuli. Phosphatidylserine was inactive when added to intact oocytes, but stimulated membrane phosphorylation nearly 15-fold when added to isolated membranes. The results indicate a link between phospholipid methylation and protein kinase C activation.

Factors affecting the response of the female rat reproductive system to cannabinoids.

Chronic oral administration of either crude marihuana extract (CME) or delta 9-tetrahydrocannabinol (THC) to female Fischer rats for 64-72 days, at a dose approximating heavy usage by humans, reduces food intake by about 8%. Pair-feeding studies demonstrate that this decreased food intake accounts for previously described decreases in uterine and ovarian weights, which are much more affected by food restriction than is body weight. THC-treated rats lost weight initially which was not regained. Pair-fed rats gained only about one-half of the weight of the untreated control or vehicle-treated control rats over a 64-day period. Although long-term cannabinoid administration leads to tolerance and the resumption of the estrous cycle, the onset of estrus is often delayed when cannabinoid is administered 5-6 hr before the proestrus luteinizing hormone (LH) surge. Our results indicate that although chronic exposure to cannabinoids can continue to affect the rat estrous cycle, they do not have a direct effect on growth of the reproductive organs. The results reemphasize the need for adequate nutritional controls in marihuana and other toxicological research.

Progesterone induction of phospholipid methylation and arachidonic acid turnover during the first meiotic division in amphibian oocytes.

Progesterone is the physiological stimulus that acts at the amphibian oocyte plasma membrane to induce the meiotic divisions. Rana oocytes were preincubated with [3H]-arachidonic acid, [3H]-methionine and/or [14C]choline. Total and plasma membrane phospholipids were monitored during the first 2 h after induction with progesterone. A transient increase in methylation of phosphatidylethanolamine during the first 10 minutes coincided with an increased Ca2+ efflux and was followed by increased arachidonic acid incorporation into phosphatidylcholine during a period of increasing membrane conductance. The labeled phospholipids disappeared sequentially 5-90 min after the hormone stimulus, suggesting that activation of phospholipases A2 and/or C occur as part of a cascade of membrane events.

NMR studies of intracellular sodium ions in amphibian oocytes, ovulated eggs, and early embryos.

23Na NMR, in combination with an anionic paramagnetic shift reagent dysprosium bis(tripolyphosphate), has been used to study intracellular Na+ in Rana oocytes, ovulated eggs, and early cleavage embryos. The technique allows accurate and simultaneous determination of both extracellular space and intracellular Na+ concentration. In prophase-arrested, follicle-enclosed oocytes, only about 17% of the total oocyte Na+ (approximately 40 mmol/kg of cells) was NMR-visible. Homogenizing oocytes in 0.24 M sucrose did not significantly affect the 23Na resonance. About 30% of the total oocyte Na+ was associated with the yolk platelets isolated at room temperature by differential centrifugation. NMR analysis, however, did not yield a detectable 23Na signal from these intact platelets. Thus, while yolk platelets are rich in Na+, this Na+ does not contribute to the oocyte 23Na NMR signal. Denuded oocytes, obtained by removing the follicular epithelium, gained about 10 mmol of total Na+/kg of cells and exhibited a comparable increase in NMR-visible Na+, suggesting the existence of compartments with varying degree of NMR visibility within the oocyte. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ with different magnetic environments and relaxation behavior in denuded oocytes. Since platelet Na+ appears to be NMR-invisible, one of the two observed compartments may be the nucleus. Progesterone action on the amphibian oocyte caused measurable changes in NMR-visible Na+. By ovulation (second metaphase), there is a gain in total egg Na+, and the NMR-visible Na+ is also increased. Following fertilization, however, there is some loss of total cell Na+ but, by the 2-4 cell stage, about 70% of the total Na+ becomes NMR-visible. These results indicate that a sizable fraction of the Na+ in follicle-enclosed, prophase oocyte is sequestered and located in NMR-invisible compartments and that changes in NMR-visible intracellular Na+ occur following hormonal and developmental stimuli.

Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques.

Insulin (0.1-10 microM) reinitiates the meiotic divisions in Rana oocytes and produces a 14-20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na+ conductance of the membrane decreases by 40-50% with a concomitant increase in 22Na+ uptake from the medium. The increased uptake of Na+ during a period of decreasing Na+ conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pHi are Na+-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li+ for Na+. Insulin hyperpolarizes Li+ or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K+-sensitive components (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [125I]Insulin binding to the plasma membrane shows a class of high-affinity sites (Kd = 87 nM) with 40-50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na+-H+ exchange resulting in an increase in intracellular pH and Na+ uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na+ conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na+ pump which together with a decrease in Na+ conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.

Effect of long-term administration of delta 9-tetrahydrocannabinol on hepatic mixed-function oxidase systems in the rat.

Chronic oral treatment of young female Fischer rats with 25 mg/kg delta 9-tetrahydrocannabinol (THC) per day inhibited aminopyrine demethylation and significantly increased benzo[a]pyrene oxidation by the liver. THC treatment also elevated serum corticosterone levels and produced a significant loss of body weight. The weight loss was not due to vehicle or food intake (pair-feeding). Pair-feeding did, however, produce a stimulation of both mixed function oxidase pathways as well as a marked elevation in serum corticosterone levels. The results indicate that THC has a differential effect on mixed function oxidase pathways in the liver that is not directly related to food intake or corticosterone levels.

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.

Endocytosis in the amphibian oocyte. Effect of insulin and progesterone on membrane and fluid internalization during the meiotic divisions.

Endocytosis has been studied in the denuded Rana pipiens oocyte using 3H-labeled inulin. Internalization of labeled inulin is linear after the first 10-15 min and uptake into the cytoplasm is temperature-dependent and is blocked by 15 microM cyanide. Uptake occurs without hydrolysis of the inulin and varies exponentially with the concentration of inulin in the medium. Based on specific activity of the medium and inulin uptake into the cytoplasm, it is estimated that a fluid volume of about 20-25 nl is internalized per oocyte per hour. This fluid phase uptake corresponds to a half-time of about 35 h for turnover of the oocyte fluid phase. An estimate of membrane area based on endocytotic vesicle size from electron micrographs suggests that the entire oocyte plasma membrane recycles several times an hour. A fraction (15-20%) of the inulin taken up is associated with the plasma-vitelline membrane complex and uptake into the membrane complex parallels uptake into the cytoplasm. Insulin (a meiotic agonist) concentrations that induce plasma membrane hyperpolarization over the first h also stimulate [3H]inulin uptake into both the oocyte cytoplasm and membrane complex over the same time period. Progesterone (the physiological inducer) has no effect on inulin uptake during the first hour, but by 16-17 h after exposure to progesterone, inulin uptake is significantly enhanced. These results suggest that hormones such as insulin and progesterone may regulate membrane permeability by a programmed internalization and possible recycling of the plasma membrane components.