Role of calcium in the bombesin-induced intestinal CCK release in rats

In the isolated vascularly perfused rat duodenojejunum, vascular infusion of bombesin (100 nM) provoked an early, transient (6 min) release of CCK (500% of basal), followed by a sustained response (400% of basal). The calcium chelator EGTA (2 mM) reduced the early peak and abolished the second phase of CCK release. A similar variation was evoked by verapamil (10 μM), whereas diltiazem (100 μM), nifedipine (50 μM), and ω-conotoxin (100 nM) had no significant effect. It is concluded that bombesin-induced CCK release from rat intestine is dependent on the availability of extracellular calcium and on the activation of calcium channels sensitive to blockers of the phenylalkylamine family.     

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid,an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca²⁺), mPB1^- (modified PBS without Ca²⁺), mPB1⁺/EGTA (mPB1⁺ containing EGTA), mPB1^-/EGTA (mPB1^- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells.

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA. These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Role of calcium in histamine release from rat mast cells activated by various secretagogues; intracellular calcium mobilization correlates with histamine release

Anti-IgE, con A or antigen caused an increase in the intracellular calcium concentration, [Ca2+]i, of mast cells. The increase occurred in two stages: a rapid initial rise caused by Ca-mobilization from intracellular Ca-stores and a second sustained rise caused by an influx of extracellular calcium (White, J.R., Pluznik, D.V., Ishizaka, K. & Ishizaka, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8193-8197). The rapid initial rise was followed by a release of histamine, which seemed to coincide with the second rise. A23187 and compound 48/80 induced a rapid initial rise in [Ca2+]i, followed by a gradual decrease in [Ca2+]i, GMCHA-OPhBut, a specific pH 7 tryptase inhibitor (Muramatu, M., Ito, T., Takei, M. & Endo, K. (1988) Biol. Chem. Hoppe-Seyler 369, 617-625), strongly inhibited both the initial and second rises of [Ca2+]i, as well as histamine release by these secretagogues, and its effects on the initial rise were closely correlated with those on histamine release. Addition of GMCHA-OPhBut immediately after the initial rise strongly inhibited the second rise, thereby decreasing the final [Ca2+]i. These results strongly suggested a possible involvement of pH 7 tryptase, not only in Ca-mobilization leading to the initial rise in [Ca2+]i, but also in the second rise. Trapping of extracellular calcium by 3mM EGTA decreased both the initial rise in [Ca2+]i and histamine secretion induced by anti-IgE or con A; the magnitude of this effect depended on the time between induction and EGTA addition. Histamine release was closely correlated with the initial rise in [Ca2+]i. Similar results were obtained with A23187, but even 5 min after the addition of EGTA an initial rise of [Ca2+]i could still be induced, and histamine (30% of total histamine) was still released. However, A23187 did not induce a rise in [Ca2+]i in mast cells which had been exhaustively washed with Tyrode/Hepes solution containing 3mM EGTA, followed by suspension in the same solution. Even at 20 min after depletion of the extracellular calcium, compound 48/80 still caused an initial rise in [Ca2+]i to above half the maximal value, and histamine secretion was even less affected. The above results indicated that the initial rise in [Ca2+]i, due to Ca-mobilization, correlates with the histamine release promoted by the secretagogues described. On the other hand, isoproterenol strongly induced histamine secretion with no change of [Ca2+]i, while EGTA treatment prior to isoproterenol stimulation had no effect on histamine release, indicating a different secretion mechanism.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Extracellular calcium and the inotropic effect of epinephrine on frog skeletal muscle

The purpose of these experiments was to determine if extracellular calcium plays an important role in mediating the inotropic effect of epinephrine in isolated frog sartorius muscle. Initial experiments indicated that epinephrine potentiated the muscle twitch in a concentration-dependent manner with concentrations of 10 microM to 1 mM, increasing peak tension by approximately 33%. To inhibit the influx of extracellular calcium, muscles were incubated for 20 min in media containing epinephrine in which calcium had been removed and replaced by magnesium or EDTA, or in experimental media containing epinephrine and the calcium channel blockers D-600 or diltiazem (5 microM). Each experimental condition was found to antagonize the effects of epinephrine such that peak twitch tensions were not significantly different from the control. When muscles were returned to normal Ringer's solution containing epinephrine, twitches exhibited progressive potentiation. Muscles were also incubated for 20 min in epinephrine without stimulation. Once stimulation was resumed, twitches were not immediately potentiated but rather gradually increased over time. These results suggest that the inotropic effects of epinephrine are influenced by the influx of extracellular calcium, an event that is dependent on muscle activation.     

Decay of the slow calcium current in twitch muscle fibers of the frog is influenced by intracellular EGTA

The mechanism(s) of the decay of slow calcium current (ICa) in cut twitch skeletal muscle fibers of the frog were studied in voltage-clamp experiments using the double vaseline-gap technique. ICa decay followed a single exponential in 10 mM external Ca2+ and 20 mM internal EGTA solutions in all pulse protocols tested: single depolarizing pulses (activation protocol), two pulses (inactivation protocol), and during a long pulse preceded by a short prepulse (400 ms) to 80 mV (tail protocol). In single pulses the rate constant of ICa decay was approximately 0.75 s-1 at 0 mV and became faster with larger depolarizations. ICa had different amplitudes during the second pulses of the inactivation protocol (0 mV) and of the tail protocol (-20 to 40 mV) and had similar time constants of decay. The time constant of decay did not change significantly at each potential after replacing 10 mM Ca2+ with a Ca2+-buffered solution with malate. With 70 mM intracellular EGTA and 10 mM external Ca2+ solutions, ICa also decayed with a single-exponential curve, but it was about four times faster (approximately 3.5 s-1 at 0 mV pulse). In these solutions the rate constant showed a direct relationship with ICa amplitude at different potentials. With 70 mM EGTA, replacing the external 10 mM Ca2+ solution with the Ca2+-buffered solution caused the decay of ICa to become slower and to have the same relationship with membrane potential and ICa amplitude as in fibers with 20 mM EGTA internal solution. The mechanism of ICa decay depends on the intracellular EGTA concentration: (a) internal EGTA (both 20 and 70 mM) significantly reduces the voltage dependence of the inactivation process and (b) 70 mM EGTA dramatically increases the rate of tubular calcium depletion during the flow of ICa.     

Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation

There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca²⁺]_i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca²⁺]_i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y₂ receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca²⁺-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y₂ receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.     

Effects of EGTA and slow freezing of bovine oocytes on post‐thaw development in vitro

Experiments were conducted to assess the morphological viability and in vitro developmental potential of bovine oocytes after exposure to Ethylene Glycol‐bis(‐aminoethyl Ether) N,N,N,N‐Tetra‐acetic Acid (EGTA) prior to slow freezing. Different concentrations of EGTA (0, 1, 5 and 10 mM) and exposure intervals (5, 10 and 15 min) were tested on immature (GV) and in vitro matured (IVM) oocytes equilibrated in 1.5 mM propylene glycol (PG) without (experiment 1) or with slow freezing (experiment 2). In addition, PG and ethylene glycol (EG) were compared for cryoprotective efficacy. In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed in defined conditions. Pretreatment of both types of oocytes with 1 mM EGTA for 5 min without freezing yielded morphological and functional results comparable to those obtained for controls while results from higher concentrations of EGTA were lower (P < 0.05). Higher rates of freeze‐thaw survival and embryonic development were obtained after pretreating GV oocytes with 1 or 5 mM EGTA for 5 min. Similarly, better results were obtained when IVM oocytes were pretreated with 1 mM EGTA for either 5 or 10 min. When pretreated with 1 mM EGTA for 5 min and frozen with PG IVM oocytes exhibited higher survival rates (P < 0.05) than those frozen with EG. However, no significant differences were observed in the in vitro development of surviving GV or IVM oocytes frozen with either PG or EG. Results suggest that a prefreeze treatment with 1 mM EGTA for 5 min can enhance oocyte viability. Conditions described enabled blastocyst development of 2.9% of GV oocytes and 8.0% of IVM oocytes after cryopreservation and IVF. Mol. Reprod. Dev. 52:86–98, 1999. © 1999 Wiley‐Liss, Inc.     

Calmodulin is involved in heat shock signal transduction in wheat

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.     

Procedural improvements for in vitro production of viable uterine stage caprine embryos

Efforts to improve proportions of caprine immature oocytes developing into viable uterine-stage embryos in vitro involved study of 1924 oocytes in experiments designed to examine influences of fertilization media, sperm incubation temperatures, sperm treatment procedures, different protein supplementations, and different insemination intervals. Oocytecumulus complexes (OCCs) were matured during 27 h in TCM-199 supplemented with 20% FBS, 100 μg LH ml⁻¹, 0.5 μg FSH ml⁻¹, and 1 μg Estradiol-17-β ml⁻¹at 38.5 °C in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere. Freshly collected sperm were washed and incubated at either 22 °C or 38.5 °C for 5 h and then treated with either 0.1 μM calcium ionophore A23187 for 1 min, or with 7.35 mM calcium lactate in the presence of oocytes during the insemination interval, or with 100 μg heparin +2 mM caffeine ml⁻¹ for 15 min. The interval for insemination was experimentally varied i.e. 14 or 24 h. Results showed that: (a) when used as a fertilization medium mDM supported more blastocyst development than TALP (10.5% vs. 0%, P < 0.05); (b) incubation temperatures of 22 °C or 38.5 °C prepared goat spermatozoa equally for capacitation in mDM containing 20% FBS; (c) when oocytes were inseminated with sperm incubated in mDM with 20% FBS and capacitated with calcium lactate more embryos reached the blastocyst stage (P < 0.05) than after incubation in the same conditions but after sperm capacitation with heparin, and A23187 (31.8% vs. 24.2% and 10.2%, respectively; (d) a 24 h insemination interval was not superior to 14 h when sperm were incubated with either 20% FBS or 6 mg BSA ml⁻¹ and capacitated with calcium lactate (P > 0.05). Three morulae resulting from the best conditions in this work (FBS, calcium lactate, 14 h insemination) were transferred into the uterine horn ipsilateral to the corpus luteum of a recipient and two normal female kids were born after normal gestation. This is the first report in which it has been possible to consistently take caprine development to the blastocyst stage in vitro, and to obtain offspring following uterine transfer. Methodology reported here should facilitate implementation of new reproductive and genetic strategies in goat breeding.     

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa)

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 M indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl₂) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA-or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.     

Skeletal muscle sarcolemma in malignant hyperthermia: evidence for a defect in calcium regulation

Sarcolemmal properties implicated in the skeletal muscle disorder, malignant hyperthermia (MH), were examined using sarcolemma-membrane vesicles isolated from normal and MH-susceptible (MHS) porcine skeletal muscle. MHS and normal sarcolemma did not differ in the distribution of the major proteins, cholesterol or phospholipid content, vesicle size and sidedness, (Na+ + K+)-ATPase activity, ouabain binding, or adenylate cyclase activity (total and isoproterenol sensitivity). The regulation of the initial rates of MHS and normal sarcolemmal ATP-dependent calcium transport (calcium uptake after 1 min) by Ca2+ (K1/2 = 0.64-0.81 microM), calmodulin, and cAMP-dependent protein kinase were similar. However, when sarcolemmal calcium content was measured at either 2 or 20 min after the initiation of active calcium transport, a significant difference between MHS and normal sarcolemmal calcium uptake became apparent, with MHS sarcolemma accumulating approximately 25% less calcium than normal sarcolemma. Calcium transport by MHS and normal sarcolemma, at 2 or 20 min, had a similar calmodulin dependence (C1/2 = 150 nM), and was stimulated to a similar extent by cAMP-dependent protein kinase or calmodulin. Halothane inhibited MHS and normal sarcolemmal active calcium uptake in a similar fashion (half-maximal inhibition at 10 mM halothane), while dantrolene (30 microM) and nitrendipine (1 microM) had little effect on either MHS or normal sarcolemmal calcium transport. After 20 min of ATP-supported calcium uptake, 2 mM EGTA plus 10 microM sodium orthovanadate were added to initiate sarcolemmal calcium efflux. Following an initial rapid phase of calcium release, an extended slow phase of calcium efflux (k = 0.012 min-1) was similar for both MHS and normal sarcolemma vesicles. We conclude that although a number of sarcolemmal properties, including passive calcium permeability, are normal in MH, a small but significant defect in MHS sarcolemmal ATP-dependent calcium transport may contribute to the abnormal calcium homeostasis and altered contractile properties of MHS skeletal muscle.     

Fatigue and contraction of slow and fast muscles in hypokinetic/hypodynamic rats

This investigation examined the effects of hypokinesia/hypodynamia (H/H) on fatigability and contractile properties of rat soleus (S) and gastrocnemius (G) muscles. Whole-body suspension for 1 wk was used to eliminate hindlimb load-bearing functions and simultaneously permit voluntary isotonic contractions. Train stimulations (45/min, 16 min) resulted in significantly (P less than 0.05) faster rates of fatigue to lower asymptotes in G from H/H rats. Fatigue in the S was minimal at this stimulation frequency and differences between H/H and control animals were not significant. Contractile properties (twitch and tetanic) were measured before and after train stimulations. H/H suspension resulted in an increased twitch tension in G. However, H/H did not change train or tetanic tensions per gram or other G contractile properties. Peak twitch, train, and tetanic tensions, time to peak tension, one-half relaxation time, and twitch and tetanic peak rates of tension development and decline were unchanged by H/H in S muscles. These results indicate that 1 wk of H/H-induced muscle atrophy significantly increases fatigability in G but does not effect contractile properties of fast-twitch (G) or slow-twitch (S) muscles.     

Association of the Igamma and Idelta charge movement with calcium release in frog skeletal muscle

Charge movement and calcium transient were measured simultaneously in stretched frog cut twitch fibers under voltage clamp, with the internal solution containing 20 mM EGTA plus added calcium and antipyrylazo III. When the nominal free [Ca2+]i was 10 nM, the shape of the broad I(gamma) hump in the ON segments of charge movement traces remained invariant when the calcium release rate was greatly diminished. When the nominal free [Ca2+]i was 50 nM, which was close to the physiological level, the I(gamma) humps were accelerated and a slow calcium-dependent I(delta) component (or state) was generated. The peak of ON I(delta) synchronized perfectly with the peak of the calcium release rate whereas the slow decay of ON I(delta) followed the same time course as the decay of calcium release rate. Suppression of calcium release by TMB-8 reduced the amount of Q(delta) concomitantly but not completely, and the effects were partially reversible. The same simultaneous suppression effects were achieved by depleting the sarcoplasmic reticulum calcium store with repetitive stimulation. The results suggest that the mobility of Q(delta) needs to be primed by a physiological level of resting myoplasmic Ca2+. Once the priming is completed, more I(delta) is mobilized by the released Ca2+ during depolarization.     

Down-regulation of MEK/ERK signaling by E-cadherin-dependent PI3K/Akt pathway in differentiating intestinal epithelial cells

In vitro experiments have shown that the establishment of cell-cell contacts in intestinal epithelial cell cultures is a critical step in initiating ERK inhibition, cell cycle arrest, and induction of the differentiation process. Herein, we determined the mechanisms through which E-cadherin-mediated cell-cell contacts modulate the ERK pathway in intestinal epithelial cells. We report that: (1) removal of calcium from the culture medium of newly confluent Caco-2/15 cells (30 min, 4 mM EGTA) results in the disruption of both adherens and tight junctions and clearly decreases Akt phosphorylation while increasing MEK and ERK activities. Akt, MEK, and ERK activation levels return to control levels 60 min after calcium restoration; (2) the use of E-cadherin blocking antibodies efficiently prevents Akt phosphorylation and MEK-ERK inhibition after 70 min of calcium restoration; (3) using the PI3K inhibitor LY294002 (15 microM) in calcium switch experiments, we demonstrate that the assembly of adherens junctions activates Akt activity and triggers the inhibition of ERK1/2 activities in a PI3K-dependent manner; (4) adenoviral infection of confluent Caco-2/15 cells with a constitutively active mutant of Akt1 strongly represses ERK1/2 activities; (5) inhibition of PI3K abolishes Akt activity but leads to a rapid and sustained activation of the MEK-ERK1/2 in confluent differentiating Caco-2/15 cells, but not in undifferentiated growing Caco-2/15 cells. Our data suggest that E-cadherin engagement leads to MEK/ERK inhibition in a PI3K/Akt-dependent pathway. This mechanism may account for the role of E-cadherin in proliferation/differentiation transition along the crypt-villus axis of the human intestinal epithelium.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Calcitonin increases pyruvate carboxylase activity in hepatic mitochondria of rats

The effect of calcitonin (CT) on calcium content and enzyme activity in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant increase in the content of calcium, the activity of pyruvate carboxylase, succinate dehydrogenase and ATPase 15 min after the hormone treatment. The significant increases in calcium content and pyruvate carboxylase activity were also observed 30 min after CT administration, while succinate dehydrogenase and ATPase activity began to decrease. A physiological dose of CT (20 MRC mU/100 g BW) caused a marked increase in calcium content and pyruvate carboxylase activity but not succinate dehydrogenase of ATPase-activity. The removal of calcium by 10 mM EGTA washing of the mitochondria produced a remarkable reduction in pyruvate carboxylase activity increased by CT administration. The addition of calcium ion of 2.5 x 10(-2) - 2.5 x 10(1) nmoles Ca2+ per mg mitochondrial protein produced a marked increase in pyruvate carboxylase activity. The present results suggest that calcium taken up by the hepatic mitochondria after CT administration activates pyruvate carboxylase.     

Calcium effects on frog retinal cyclic guanosine 3’,5’- monophosphate levels and their light-initiated rate of decay

When retinal sections were isolated from dark-adapted bullfrogs and placed in normal ringer’s solution, they contained 40.7 +/- 0.2 pmol cGMP/mg protein (mean +/- SEM, 30 samples). When isolated, dark-adapted retinal sections were removed from normal ringer’s solution and placed in calcium-deficient ringer’s solution with 3 mM EGTA, there was about a threefold rise in cyclic GMP (cGMP) levels by 1.5 min and about a 10-fold rise by 5 min. The cGMP level remained high with no detectable decrease for at least 40 min (the longest time measured). When isolated, dark- adapted retinal sections were removed from normal ringer’s solution and placed in ringer’s solution which contained high- calcium (20 mM CaCl(2)), there was a slow but significant decrease in cGMP levels. After 20 min in high-calcium ringer’s solution the cGMP level was 0.58 +/- 0.07 (mean +/- SEM, eight samples) of the cGMP level in normal ringer’s solution incubated for the same time. The rate at which 10-fold elevated cGMP levels in low calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium decreased upon illumination was examined using quick-freezing techniques on the retinal sections. The elevated cGMP level in retinal sections incubated in low-calcium ringer’s solution was found to decay about 15-fold faster than cGMP levels in retinal sections incubated in normal ringer’s solution. The CGMP level in low calcium was significantly different (P=0.005) after 1 s illumination, whereas the cGMP level in normal calcium was not significantly different.