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Cytosolic calcium oscillators.
Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.
ADP-ribose gates the fertilization channel in ascidian oocytes.
We report an ion channel in the plasma membrane of unfertilized oocytes of the ascidian Ciona intestinalis that is directly gated by the second messenger ADP-ribose. The ion channel is permeable to Ca2+ and Na+ and is characterized by a reversal potential between 0 and +20 mV and a unitary conductance of 140 pS. Preinjection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or antagonists of intracellular Ca2+ release channels into oocytes did not inhibit the ADP-ribose current, demonstrating that the channel is activated in a Ca2+-independent manner. Both the fertilization current and the current induced by the injection of nicotinamide nucleotides are blocked by nicotinamide, suggesting that the ADP-ribose channel is activated at fertilization in a nicotinamide-sensitive manner. These data suggest that ascidian sperm trigger the hydrolysis of nicotinamide nucleotides in the oocyte to ADP-ribose and that this mechanism is responsible for the production of the fertilization current.
Actions of cADP-ribose and its antagonists on contraction in guinea pig isolated ventricular myocytes. Influence of temperature.
Although it is becoming widely accepted that cADP-ribose (cADPR) can regulate calcium release from the endoplasmic reticulum in sea urchin eggs and in a variety of mammalian cell types, it remains controversial whether this substance might influence calcium release during excitation-contraction coupling in cardiac muscle. We have investigated possible actions of cADPR in intact cells isolated from guinea pig ventricle, paying particular attention to the possible influence of temperature. At 36 degrees C, myocyte contraction was influenced by cytosolic application of cADPR in a concentration-dependent manner (showing an approximately 30% increase in contraction with 5 mumol/L cADPR applied via a patch pipette in myocytes stimulated to fire action potentials at 1 Hz). Calcium transients measured with fura 2 were also increased by 5 mumol/L cADPR. Antagonists of cADPR reduced contraction at 36 degrees C (by approximately 35% with either 50 mumol/L 8-Br-cADPR or 5 mumol/L 8-amino-cADPR applied via the patch pipette). At room temperature (approximately 20 degrees C to 24 degrees C), no significant effects on contraction were detected with either cADPR or its antagonists. At 36 degrees C, treatment of the cells with a mixture of 2 mumol/L ryanodine and 1 mumol/L thapsigargin to suppress function of the sarcoplasmic reticulum stores of calcium prevented the action of 5 mumol/L cADPR applied via a patch pipette. These observations are consistent with an action of cytosolic cADPR to enhance calcium-induced calcium release from the sarcoplasmic reticulum in guinea pig ventricular myocytes at 36 degrees C. The observed influence of temperature under the conditions of our experiments is one factor that might help to account for failure to detect actions of cADPR and its analogues in some previous studies.
Mechanisms of calcium release and sequestration in eggs of Chaetopterus pergamentaceus.
Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.
Effects of glyburide-cyclosporin A interaction on interleukin-2 production in rats.
The effects of simultaneous administrations of Cyclosporin A (CsA) and Glyburide on the immune system of rats has been evaluated in terms of Interleukin-2 (IL-2) production by Concanavalin A (ConA) stimulated splenocytes and exogenous IL-2 binding capacity. The inhibitory effect of Cyclosporin A on IL-2 production of lymphoid cells is well known. Spleen cells from rats receiving CsA had reduced levels of IL-2 when compared to untreated controls or rats receiving Glyburide only. Splenocytes from rats receiving both drugs had reduced levels of IL-2 when they were sacrificed 24 hours after one or three CsA administrations; instead when the animals were sacrificed 6 days after three CsA administrations, their ability of producing IL-2 is increased as well as increasing exogenous IL-2 binding capacity. These findings let us hypothesize that when there are lower concentrations of CsA in lymphocytes there is an increase of cellular metabolism induced by Glyburide that leads to an increase in IL-2 secretion and in IL-2 receptor expression on cellular surface restoring these levels to normal or slightly above normal levels.
Pharmacological properties of the Ca2+-release mechanism sensitive to NAADP in the sea urchin egg.
1. The sea urchin egg homogenate is an ideal model to characterize Ca2+-release mechanisms because of its reliability and high signal-to-noise-ratio. Apart from the InsP3- and ryanodine-sensitive Ca2+-release mechanisms, it has been recently demonstrated that this model is responsive to a third independent mechanism, that has the pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP), as an endogenous agonist. 2. The sea urchin egg homogenate was used to characterize the pharmacological and biochemical characteristics of the novel Ca2+-releasing agent, NAADP, compared to inositol trisphosphate (InsP3) and cyclic ADP ribose (cyclic ADPR), an endogenous activator of ryanodine receptors. 3. NAADP-induced Ca2+-release was blocked by L-type Ca2+-channel blockers and by Bay K 8644, while InsP3- and cyclic ADPR-induced Ca2+-release were insensitive to these agents. L-type Ca2+-channel blockers did not displace [32P]-NAADP binding, suggesting that their binding site was different. Moreover, stopped-flow kinetic studies revealed that these agents blocked NAADP in a all-or-none fashion. 4. Similarly, a number of K+-channel antagonists blocked NAADP-induced Ca2+-release selectively over InsP3- and cyclic ADPR-induced Ca2+-release. Radioligand studies showed that these agents were not competitive antagonists. 5. As has been shown for InsP3 and ryanodine receptors, NAADP receptors were sensitive to calmodulin antagonists, suggesting that this protein could be a common regulatory feature of intracellular Ca2+-release mechanisms. 6. The presence of K+ was not essential for NAADP-induced Ca2+-release, since substitution of K+ with other monovalent cations in the experimental media did not significantly alter Ca2+ release by NAADP. On the contrary, cyclic ADPR and InsP3-sensitive mechanisms were affected profoundly, although to a different extent depending on the monovalent cation which substituted for K+. Similarly, modifications of the pH in the experimental media from 7.2 to 6.7 or 8.0 only slightly affected NAADP-induced Ca2+-release. While the alkaline condition permitted InsP3 and cyclic ADPR-induced Ca2+-release, the acidic condition completely hampered both Ca2+-release mechanisms. 7. The present results characterize pharmacologically and biochemically the novel Ca2+-release mechanism sensitive to NAADP. Such characterization will help future research aimed at understanding the role of NAADP in mammalian systems.
The relative order of IP3 sensitivity of types 1 and 3 IP3 receptors is pH dependent.
The type-3 inositol 1,4,5-trisphosphate (IP3) receptor, in contrast to the type-1 IP3 receptor (IP3R), is not stimulated by sulfhydryl oxidation and is less sensitive to adenosine 5'-triphosphate. In the present study we compared the effect of pH on the Ca2+ release induced by IP3 and cytosolic Ca2+ between IP3R3-expressing 16HBE14o- cells and IP3R1-expressing A7r5 cells. Changing pH from 6.8 to 7.5 decreased the IP3 concentration required for half-maximal stimulation of IP3R3 (EC50) 10.7-fold (from 2.14 to 0.20 microM). Similar alkalinization decreased the IP3 concentration (EC50) for stimulation of IP3R1 only 2.5-fold (from 0.87 to 0.35 microM). IP3R1 is therefore the more sensitive isoform at pH 6.8, while IP3R3 is more sensitive at pH 7.5. Stimulation and inhibition of IP3R1 and -3 by low and high cytosolic [Ca2+] respectively was observed at both pH 6.8 and 7.5. Increasing [H+] shifted the Ca2+-activation curve of IP3R1 towards higher [Ca2+] but did not affect the Ca2+ dependence of IP3R3. We conclude that IP3R1 and -3 differ markedly in their response to protons.
Modulation of Xenopus embryo mesoderm-specific gene expression and dorsoanterior patterning by receptors that activate the phosphatidylinositol cycle signal transduction pathway.
A role for the phosphatidylinositol (PI) cycle signal transduction pathway in Xenopus mesoderm induction has been revealed by observations of PI cycle activation coincident with this process, combined with the demonstration that Li+ (a PI cycle inhibitor) blocks this response and hyperdorsalizes mesoderm induction in intact embryos or augments growth factor-mediated induction in animal caps. It has been suggested that spatially restricted PI cycle activity in the marginal zone might modulate (but not, itself, activate) mesoderm induction. To better characterize the ability of PI cycle activity to modulate the pattern of mesoderm-specific gene expression elicited by mesoderm-inducing growth factors we have expressed in the embryo exogenous 5-hydroxytryptamine receptors that activate the PI cycle. In embryos, ventral expression and activation of these receptors during mesoderm induction are without obvious effect, whereas dorsal expression and activation yield dorsoanterior-deficient tadpoles. In animal caps induced with activin, simultaneous activation of exogenous 5-hydroxytryptamine receptors inhibits both convergent extension movements associated with dorsal mesoderm induction and the expression of goosecoid, a dorsal-specific gene, but is without effect on expression of a 149 generic mesodermal marker, Xbra. All of these effects of a 149 PI cycle-stimulating receptor are the opposites of those previously reported for the PI cycle inhibitor, Li+. PI cycle activity thus proves able to modulate the dorsal/ventral character of early mesodermal gene expression elicited by growth factor, suggesting a model for mesodermal patterning.
Lithium-induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells.
1. Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence. 2. We have reported previously that treatment of GH3 cells with 1 mM Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin-releasing hormone (TRH)-stimulated levels of mass inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected. 3. The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mM Li+ for 7 days relative to control cells. 4. The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower 'basal' [Ca2+]i in a population of cells and to inhibit agonist-stimulated increases in [Ca2+]i. Under these conditions, basal and TRH-stimulated mass Ins(1,4,5)P3 levels were unaffected. 5. These results demonstrate that a 7-day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.
[The action of cyclosporin A (CSA) on glycogen synthesis processes in rats in vivo].
Cyclosporin A appears to act on type I diabetes. In rats it alters glucose digestion. The present study provides a more detailed analysis of the influence of this substance on certain aspects of carbohydrate metabolism. The data reveal that CSA increases the glycogen in the liver and muscle but reduces the amount in the heart. Blood sugar was also significantly increased but only after the third dose of CSA. While these preliminary results clearly show that CSA has an effect on carbohydrate metabolism they do not indicate whether is the drug itself or one of the hormones that usually influence carbohydrate exchange which has this effect.
Influence of haemodialysis on lipase activity.
Our aim was to determine whether the increase in serum pancreatic lipase values, reported in patients with chronic renal failure maintained on haemodialysis, is the result of haemoconcentration by fluid removal during dialysis, or whether it is due to lipase stimulation by endothelial lipoprotein lipase, induced by the heparin used as an anticoagulant. We therefore compared the increases in serum lipase, when heparin was used, with those observed when this was replaced by the antithrombotic agent, defibrotide, which has no effect on lipoprotein lipase. In addition, in order to determine the effects of haemoconcentration, variations in total protein concentration and haematocrit values were determined on the same samples, both before and after dialysis. The results showed a statistically significant post-dialysis increase in lipase only when heparin was used (p < 0.03). There was also a mean percentage post-dialysis increase of 16.2% in total protein (p < 0.0001) and 15.5% in haematocrit (p < 0.0001), due to fluid removal. No significant correlation in percentage increases was found between lipase vs total protein or haematocrit values. These findings suggest that heparin-induced lipoprotein lipase stimulation is the principal cause of the post-dialysis increase in pancreatic lipase, and that fluid removal during dialysis makes only a minor contribution to this increase.
Cyclic ADP-ribose, the ADP-ribosyl cyclase pathway and calcium signalling.
Cyclic adenosine diphosphate-ribose, an endogenous metabolite of nicotinamide adenine dinucleotide was first characterized as a potent Ca2+ mobilizing agent in sea urchin eggs. Mounting evidence points to it being an endogenous activator of Ca(2+)-induced Ca2+ release by non-skeletal muscle ryanodine receptors in several invertebrate and mammalian cell types. Cyclic adenosine diphosphate-ribose is synthesized by adenosine diphosphate-ribosyl cyclases, which have been found to be widespread enzymes. Recent data suggests that cyclic adenosine diphosphate-ribose may function as a second messenger in sea urchin eggs at fertilization and in stimulus secretion coupling in pancreatic beta-cells. A second messenger role for cyclic adenosine diphosphate-ribose requires that its intracellular levels be under the control of extracellular stimuli. Another second messenger, cGMP, stimulates the synthesis of cyclic adenosine diphosphate-ribose from nicotinamide adenine dinucleotide by activating the adenosine diphosphate-ribosyl cyclase pathway in sera urchin eggs and egg homogenates, suggesting that cyclic adenosine diphosphate-ribose may be an intracellular messenger for cell surface receptors or nitric oxide, which activate cGMP-producing guanylate cyclases. Cyclic adenosine diphosphate-ribose may have a similar role to inositol trisphosphate in controlling intracellular calcium signalling with these two calcium-mobilizing second messengers activating ryanodine receptors and inositol trisphosphate receptors respectively.
Differential effect of temperature on histamine- and carbachol-stimulated inositol phospholipid breakdown in slices of guinea-pig cerebral cortex.
Slices of guinea-pig cerebral cortex were incubated with [3H]-inositol at 37 degrees C before exposure to histamine or carbachol at 37 degrees C or 25 degrees C. Histamine-stimulated accumulation of [3H]-inositol 1-phosphate ([3H]-IP1) at 25 degrees C was only 5-7% of that at 37 degrees C, whereas for carbachol the response at 25 degrees C was 45-49% of that at 37 degrees C. The affinity of benzilylcholine, obtained from inhibition of carbachol-induced accumulation of [3H]-IP1 was similar at 25 degrees C and 37 degrees C, but the EC50 for carbachol was lower at 25 degrees C (20 +/- 2 microM) than at 37 degrees C (42 +/- 2 microM). The IC50 for histamine inhibition of [3H]-mepyramine binding to homogenates of guinea-pig cerebral cortex did not differ significantly at 25 degrees C and 37 degrees C. Histamine-induced accumulations of [3H]-IP2 and [3H]-IP3 at 25 degrees C, expressed as a percentage of the accumulation at 37 degrees C, were also much less than the corresponding value for carbachol. These observations imply that the locus or pathway(s) of agonist-induced formation of [3H]-IP1 are not the same for histamine and carbachol.