The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

Muscarinic acetylcholine receptors of the chick amnion

The presence of muscarinic (M) acetylcholine receptors in the noninnervated chick amnion makes it possible to analyze their functioning with presynaptic effects excluded. The M receptors of the amnion mediating its contraction were identified by testing with selective antagonists: pirenzepine for M₁, methoctramine for M₂, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) for M₃, and tropicamide for M₄ receptor subtype. All antagonists acted as competitive inhibitors of M-acetylcholine receptors. With respect to cholinolytic activity estimated from the response to carbacholine (CBC) (-logIC₅₀), the antagonists could be arranged in the following series: 4-DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > methoctramine (5.63). In addition, the effect of forskolin (5 μM), activator of adenylate cyclase (AC), was unidirectional with ?-adrenergic agonists; it blocked CBC-induced contractile activity of the amnion, whereas phospholipase C (1.25 U/ml) stimulated this activity. These data suggest that CBC-or acetylcholine (ACh)-induced contractile activity of the amnion is mediated by M₃ acetylcholine receptors. Evaluation of contractile response to ACh by the tonic component usually revealed one pool of M₃ acetylcholine receptors. One pool was also revealed after treatment with 4-DAMP, with the Hill coefficient being increased (ACh, n = 1.07; ACh against the 4-DAMP background, n = 1.48). It is possible to detect two pools of M₃-acetylcholine receptors on the basis of either phase-frequency or tonic response, i.e., independently of the test parameter.     

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca²⁺ inside the ER ([Ca²⁺]_ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca²⁺]_ER (EC₅₀) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca²⁺]^ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 μM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 μM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca²⁺. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 107–112, March–April, 2007.     

Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

A Decrease of neuroprotective effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A₂ (bromophenacyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effects by ganglioside GM1 amounted to 52.8 ± 4.2%. However, in the presence in the medium of 0.1 and 1 μM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 ± 6.5% and 11.7 ± 9.8%, respectively. GM1 prevented Na⁺,K⁺-ATPase oxidative inactivation produced by H₂O₂, but in the presence of 1 μM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases-genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 μM, whereas at a higher concentration 10 μM, genistein depressed statistically significantly the GM1 neuroprotective effect. It was found that inhibitor of phospholipase A₂ bromophenacyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252a decreases or practically prevents the ganglioside GM1 neuroprotective effect on PC12 cells under stress conditions; the same ability is characteristic of genistein—an inhibitor of tyrosine kinases of the wider spectrum of action.     

Comparison of the muscarinic-cholinergic and lysophosphatidate inhibition of fibroblast adenylate cyclase demonstrating desensitization to the cholinergic stimulus

Muscarinic stimulation of cultured fibroblasts decreases initial rates of cAMP accumulation in response to hormones 50–70%. This inhibitory effect of muscarinic stimulation on cAMP accumulation in intact cells was desensitized 65–75% by a 60 min pretreatment with the muscarinic agonist carbachol (10 μM), with a $\text{t}\text{1}\text{2}$ of 11 min. The carbachol pretreatment resulted in a diminished carbachol inhibition of adenylate cyclase in broken cell preparations. The phospholipid monooleylphosphatidate (MOPA) which also inhibited hormone-stimulated cAMP accumulation with a half maximal effect at 0.03 μM (as compared with 0.5 μM for carbachol), displayed many of the characteristics of muscarinic inhibition such as loss of activity with time of pretreatment. However, fibroblasts did not become desensitized to prolonged MOPA treatment; rather, it appeared that the MOPA was being inactivated. Also, the desensitization to carbachol did not prevent further inhibition by MOPA. The inhibitory effects of maximal doses of MOPA and carbachol in combination were no greater than the effect of carbachol alone, suggesting that they shared an intermediate in their inhibition of cAMP accumulation. These results are consistent with the hypothesis that muscarinic inhibition of adenylate cyclase is mediated by the formation of a phospholipid. However, the desensitization to the cholinergic stimulus does not appear to involve the intermediate, but rather a modification at the receptor level.     

Effects of mepacrine on uterine contractile responses

Mepacrine is a potent inhibitor of uterine contractile responses in vitro. Pretreatment of isolated rat uterine horns with mepacrine (1.3 X 10(-4)M) for periods of time ranging from 15 s to 5 min prior to the addition of carbachol (1.0 X 10(-4)M) showed that mepacrine could significantly reduce carbachol-induced uterine contractile responses within 15 s of exposure. The maximal inhibitory effects of mepacrine on uterine contractile responses were observed within 2 min of mepacrine treatment. A dose-response study related to the effect of increasing concentrations of mepacrine (7.5 X 10(-6) to 1.3 X 10(-4)M) on carbachol-induced (1 X 10(-4)M) uterine contractions revealed that a dose of 3.1 X 10(-5)M mepacrine reduced the carbachol-induced contraction by 50%. A dose of 7.8 X 10(-5)M mepacrine produced the maximal inhibitory effect on the carbachol-induced uterine contractions. Two doses of mepacrine (3.1 X 10(-5) and 1.3 X 10(-4)M) significantly reduced maximal contractile responses and shifted contractile dose-response curves of carbachol, oxytocin, prostaglandin F2 alpha, and BaCl2 to the right. Based on the nonselective inhibition by mepacrine of contractile responses induced by different uterotonic agents, these results suggest that mepacrine cannot be used to characterize the role of phospholipase in regulating the actions of hormones in uterine tissue.     

Effects of Ca2+-ATPase inhibitors, ionomycin, and pharmacological modulators of ryanodine receptor on calcium release from intracellular pools and on oscillatory contractile behavior in Physarum polycephalum

Changes in calcium levels in organelles of the plasmodium of the myxomycete Physarum polycephalum were analyzed using the fluorescent calcium indicator chlortetracycline (CTC). Both the Ca2+-ATPase inhibitor 2,5;-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (100 microM) and the calcium ionophore ionomycin (1 microM) induce a significant decrease in fluorescence level (by 30%) in CTC-stained microplasmodia; this is caused by release of calcium from intracellular storage compartments. An activator of ryanodine receptors, caffeine (10-50 mM), is less effective on Ca2+ release than BHQ or ionomycin, and their inhibitor, ryanodine (100 microM), almost completely blocks the response to caffeine, but only slightly decreases the effects of BHQ or ionomycin. Procaine, another inhibitor of ryanodine receptors, at 10 mM concentration completely abolishes both the BHQ and the ionomycin responses, but 50 mM is necessary to block the effect of 25 mM caffeine. These results suggest that both the BHQ- and the ionomycin-dependent Ca2+ releases occur through the ryanodine receptor and are to be considered as calcium-induced Ca2+ release (CICR). Both the ionomycin and the BHQ responses persist in the presence of Cd2+, which blocks Ca2+ channels of the plasmalemma. In most cases, Cd2+ itself induces release of Ca2+ from the CTC-stained calcium pool; the more effective Cd2+ is, the less the following ionomycin or BHQ responses occur. This indicates that Ca2+ entry through plasmalemma plays no significant role in the ionomycin- or BHQ-evoked initiation of CICR, and that the Cd2+- and BHQ/ionomycin-depleted Ca2+ stores overlap.     

The role of cytoplasmic calcium in the regulation of the resting potential in the pulmonary veins myocardium in rats and mice

We have demonstrated the phenomenon of Са²⁺-induced hyperpolarization in the myocardium of pulmonary veins (PVs) in rats. An increase in cytoplasmic calcium [Са²⁺]_i was shown to shift the resting potential (RP) in the PVs towards more negative values. The compounds inducing an increase in [Са²⁺]_i, such as isoproterenol (10 μM), caffeine (5 mM), and ryanodine (0.01 μM), caused hyperpolarization of 10 ± 2, 9 ± 1.3, and 4.1 ± 2 mV, respectively. The inhibition of calcium-dependent potassium currents (IK_Ca) did not change RP of PVs under the control conditions and did not affect the Са²⁺-induced hyperpolarization.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP–DPAM, SNAP–OAG and SNAP–PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP–OAG 0, SNAP–DPAM 17.4 ± 2.5% and SNAP–PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca²⁺ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP–DPAM 20.7 ± 2.6%, SNAP–OAG 16.7 ± 2.9% or SNAP–PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.     

Calcium Mobilization by Ryanodine Promotes the Phosphorylation of Initiation Factor 2α Subunit and Inhibits Protein Synthesis in Cultured Neurons

Abstract: Protein synthesis plays an important role in the viability and function of the cell. There is evidence indicating that Ca²⁺ may be a physiological regulator of the translational process. In the present study, the effect of agents that increase intracellular calcium levels by different mechanisms, as well as repercussion on the rate of protein synthesis, including phosphorylation of initiation factor 2α subunit, and double-stranded RNA-dependent eIF-2α kinase (PKR) activity were analyzed. Glutamate (100 µ M ) and K⁺ (60 m M ), which increase intracellular calcium levels (the former mostly by the influx of extracellular calcium via voltage-sensitive calcium channels, and the latter by receptor-operated calcium channels), and carbachol (1 m M ), as well as glutamate, which mobilizes intracellular calcium from the endoplasmic reticulum via activation of inositol 1,4,5-trisphosphate receptor, did not modify any of the analyzed parameters. Nevertheless, 100 n M ryanodine, which increases intracellular calcium concentration by activating the ryanodine receptor, promoted a significant decrease in the rate of protein synthesis and increased both initiation factor 2α subunit phosphorylation and PKR activity. From our results, we can conclude that inhibition of protein synthesis is dependent on the mobilization of intracellular calcium from internal stores. Moreover, they strongly suggest that this inhibition is only promoted when calcium is increased via ryanodine receptor, and possibly by activation of PKR activity.     

D2 receptor-mediated inhibition of dopamine release in the rat striatum in vitro is modulated by CB1 receptors: studies using fast cyclic voltammetry

Cannabinoid CB₁ receptors are highly expressed in the striatum where they are known to be co‐localized with dopamine D₂ receptors. There is now strong evidence that cannabinoids modulate dopamine release in the brain. Using fast cyclic voltammetry, single pulse stimulation (0.1 ms; 10 V) was applied every 5 min and peak dopamine release was measured with a carbon fibre microelectrode. Application of the D₂ receptor agonist, quinpirole, inhibited single pulse dopamine overflow in a concentration‐dependent manner (IC₅₀: 3.25 × 10^?8 M). The CB₁ receptor agonist WIN55212‐2 (WIN; 1 μM) had no effect on single pulse dopamine release (93.9 ± 6.6% at 60 min, n = 5) but attenuated the inhibitory effect of quinpirole (30 nM; quinpirole 39.0 ± 4.2% vs. quinpirole + WIN, 48.2 ± 3.7%, n = 5, p < 0.05). This affect was antagonized by the CB₁ receptor anatgonist [N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide] (AM‐251, 1 μM). Dopamine release evoked by four pulses delivered at 1 Hz (4P1Hz) and 10 pulses delivered at 5 Hz (10P5Hz) was significantly inhibited by WIN [72.3 ± 7.9% control (peak 4 to 1 ratio measurement) and 66.9 ± 3.8% control (area under the curve measurement), respectively, p < 0.05; n = 6 for both]. Prior perfusion of WIN significantly attenuated the effects of quinpirole on multiple pulse‐evoked dopamine release (4P1Hz: quinpirole, 28.4 ± 4.8% vs. WIN + quinpirole, 52.3 ± 1.2%; 10P5Hz: quinpirole, 29.5 ± 1.3% vs. WIN + quinpirole, 59.4 ±7.1%; p < 0.05 for both; n = 6). These effects were also antagonized by AM‐251 (1 μM). This is the first report demonstrating a functional, antagonistic interaction between CB₁ receptors and D₂ autoreceptors in regulating rat striatal dopamine release.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Muscarinic receptors in pancreatic islets of the rat: Demonstration and dependence on long-term glucose environment

The presence of muscarinic receptors in islets of Langerhans was assessed by measurement of specific binding of [³H]methylscopolamine. Specific binding was defined as total binding minus binding obtained in the presence of 1000-fold or higher excess of unlabeled methylscopolamine. At 37°C specific binding was significant after 1 min and plateaued after 10 min of incubation. Displacement of label by increasing concentrations of unlabeled methylscopolamine indicated a dissociation constant of 1.5·10^?12 M. Effects of methylscopolamine on insulin release were evaluated from the inhibitions of cholinergic-induced insulin release. 4·10^?10 M methylscopolamine inhibited acetylcholine (20 μM)-induced insuliln release more than 60%. Binding was not influenced by the following variations during binding incubations: changing the glucose concentration from 0 to 83 mM, adding rotenon (1 μM) or omitting calcium from the incubation medium. Islets kept in tissue culture exhibited higher binding when cultured at 11.1 than at 3.3 mM glucose for 96 h. It is concluded that islets contain muscarinic receptors, the binding to which can be subject to alteration by the long-term glucose environment.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca²⁺-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca²⁺-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of ⁴⁵Ca from cells preequilibrated with ⁴⁵Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular ⁴⁵Ca level. Inositol trisphosphate, but not cAMP, evoked ⁴⁵Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Effects of α-Bungarotoxin and Reversible Cholinergic Ligands on Normal and Denervated Mammalian Skeletal Muscle

α-Bungarotoxin (BuTX; 5 μg/ml) completely blocked the endplate potential and extrajunctional acetylcholine (ACh) sensitivity of surface fibers in normal and chronically denervated mammalian muscles, respectively, in about 35 min. A 0.72 ± 0.033 mV amplitude endplate potential returned in normal muscle fibers after 6.5 hr. of washout of α-BuTX, and an ACh sensitivity of 41.02 ± 3.95 mV/nC was recorded in denervated muscle after 6.5 hr of wash (control being 1215 ± 197 mV/nC). A two-step reaction of BuTX with binding sites which may allosterically interact is postulated.

Several pharmacologic differences were noted between the ACh receptors at the normal endplate and those appearing extrajunctionally following denervation. In normal innervated muscles exposed to BuTX in the presence of 20 μM carbamylcholine or decamethonium, washout of both drugs restored twitch to control levels within 2 hr. Endplate potentials large enough to initiate action potentials were also recorded in most surface fibers. In contrast, these agents, in much higher concentrations (50 μM), were almost ineffective in preventing BuTX blockade of ACh sensitivity in denervated muscle. Hexamethonium (10 and 50 mM) depressed neuromuscular transmission and blocked the action of BuTX in normal muscle in a dose-dependent fashion. On the extrajunctional receptors, hexamethonium (50 mM) was ineffective in protecting against BuTX. We may conclude that at the normal endplate region there are two distinct populations of ACh receptors, both of which react with cholinergic ligands and BuTX, but that a small population (representing ± 1% of the total) reacts with BuTX reversibly. Our findings further suggest a clear distinction between ACh receptors located at the normal endplate region and those of the extrajunctional region of the chronically denervated mammalian muscle.     

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E₂ (PGE₂) by amnion cells. Transforming growth factor-β (TGF-β) inhibits the cytokine-stimulated PGE₂ production. In the present study, we investigated the binding of IL-1β on human amnion cells in culture. Untreated amnion cells possessed 540±60 IL-1 receptors per cell, with a dissociation constant of 1.4±0.4 nM. Cells treated with TGF-β1 (10 ng/ml) had 570±110 receptors per cell. TNF-α (50 ng/ml) increased the number of IL-1 receptors to 2930±590. TGF-β1 inhibited the receptor upregulation by TNF-α. Cells treated with TGF-β1 and TNF-α expressed 1140±590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE₂ production by IL-1β, but not by TNF-α. Amnion cells secreted large amounts of IL-1ra (1.1±0.3 ng/10⁵ cells). Treatment of the cells with TGF-β1 or TNF-α did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE₂ production.     

Role of gastrin-releasing peptide in pepsinogen secretion from the isolated perfused rat stomach

We studied the effects of the neuropeptide gastrin-releasing peptide on pepsinogen secretion using an isolated perfused rat stomach with intact vagal innervation. Following electrical stimulation of the vagus nerves, the pepsin output to the luminal effluent increased from 94 ± 7 to 182 ± 24 units pepsin/min and the release of immunoreactive gastrin-releasing peptide to the venous effluent increased from 0.059 ± 0.014 to 0.138 ± 0.028 pmol/min. Infusion of gastrin-releasing peptide at 10⁻⁸ M significantly increased pepsin output (from 87 ± 17 to 129 ± 22 units pepsin/min) and simultaneous infusion of gastrin-releasing peptide and carbachol at 10⁻⁸ and 10⁻⁶ M, respectively, resulted in an increase to almost 4 times the basal values. Atropine reduced but did not abolish the pepsin response to vagal stimulation and to infusion of gastrin-releasing peptide. Our results suggest that gastrin-releasing peptide participates in the vagal control of pepsinogen secretion.