Child protection or professional self-preservation by the baby nurses? Public health nurses and child protection in Ireland

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca2+]i) followed by a sustained rise. In Ca(2+)-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca2+]i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of -60 mV, the muscarine-induced [Ca2+]i rise, especially its sustained phase, decreased in magnitude. Intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca2+]i and inhibited the following [Ca2+]i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca2+]i. Muscarine-induced sustained [Ca2+]i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca2+]i rise and Mn2+ influx in response to muscarine were suppressed by a voltage-dependent Ca2+ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca2+ entry, but also Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca(2+)-channels may function as one of the Ca2+ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C

The effects of adrenomedullin (AM), a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2+ concentration ([Ca2+]i) at concentrations greater than 10(-7) M. Human calcitonin gene-related peptide (CGRP), a peptide structurally related to AM, also increased [Ca2+]i with a potency similar to that of AM. AM increased [Ca2+]i in the absence of extracellular Ca2+. Further, AM increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) level in a concentration-dependent manner similar to that of AM-induced [Ca2+]i, suggesting that AM-induced elevation of [Ca2+]i is due to Ca2+ release from Ins(1,4,5)P3-sensitive stores. AM (10(-9) to 10(-6) M) increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the [Ca2+]i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases [Ca2+]i through a mechanism independent of cAMP.     

Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-trisphosphate (IP3) [corrected] binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at approximately 300 nM-1 microM, the open probability remained elevated (approximately 0.8) in the presence of saturating levels (10 microM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) approximately 2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 microM and Hill coefficient (Hinh) approximately 4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.     

Fluoride activates G protein-dependent and -independent pathways in dispersed intestinal smooth muscle cells

The existence of G protein-dependent and -independent mechanisms activated by sodium fluoride was examined in muscle cells isolated separately from the circular and longitudinal layers of guinea pig intestine. The cells were transiently permeabilized by incubation with Trans. Port Reagent in the presence or absence of GDP beta S (100 microM) and then re-sealed. In the absence of GDP beta S, NaF (1 mM) induced contraction and caused an increase in [Ca2+]i, IP3 and diacylglycerol levels and in protein kinase C (PKC) activity in both cell types. In the presence of GDP beta S, the increases in IP3, DAG and PKC were abolished whereas contraction and the increase in [Ca2+]i were partly inhibited. Residual contraction and [Ca2+]i were abolished by the Ca2+ channel blocker, methoxyverapamil. We conclude that contraction and Ca2+ mobilization induced by NaF is mediated by G protein activation as well as by a G protein-independent mechanism involving activation of plasmalemmal Ca2+ channels.     

The effect of a secreted form of beta-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones

1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.     

Cooperative activation of IP3 receptors by sequential binding of IP3 and Ca2+ safeguards against spontaneous activity

BACKGROUND: Ca2+ waves allow effective delivery of intracellular Ca2+ signals to cytosolic targets. Propagation of these regenerative Ca2+ signals probably results from the activation of intracellular Ca2+ channels by the increase in cytosolic [Ca2+] that follows the opening of these channels. Such positive feedback is potentially explosive. Mechanisms that limit the spontaneous opening of intracellular Ca2+ channels are therefore likely to have evolved in parallel with the mechanism of Ca2+-induced Ca2+ release. RESULTS: Maximal rates of 45Ca2+ efflux from permeabilised hepatocytes superfused with medium in which the [Ca2+] was clamped were cooperatively stimulated by inositol 1,4,5-trisphosphate (IP3). A minimal interval of approximately 400 msec between IP3 addition and the peak rate of Ca2+ mobilisation indicate that channel opening does not immediately follow binding of IP3. Although the absolute latency of Ca2+ release was unaffected by further increasing the IP3 concentration, it was reduced by increased [Ca2+]. CONCLUSIONS: We propose that the closed conformation of the IP3 receptor is very stable and therefore minimally susceptible to spontaneous activation; at least three (probably four) IP3 molecules may be required to provide enough binding energy to drive the receptor into a stable open conformation. We suggest that a further defence from noise is provided by an extreme form of coincidence detection. Binding of IP3 to each of its four receptor subunits unmasks a site to which Ca2+ must bind before the channel can open. As IP3 binding may also initiate receptor inactivation, there may be only a narrow temporal window during which each receptor subunit must bind both of its agonists if the channel is to open rather than inactivate.     

Inhibition of bradykinin-induced calcium increase by phosphatase inhibitors in neuroblastoma x glioma hybrid NG108-15 cells

Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by bradykinin by approximately 63%. This inhibition was dependent on the concentration of okadaic acid with an IC50 of 0.15 nM. Okadaic acid treatment only lowered the maximal response of [Ca2+]i increase and had no effect on the EC50 value for bradykinin regardless of the presence of extracellular Ca2+. Neither the capacity of 45Ca2+ accumulation within intracellular nonmitochondrial Ca2+ stores nor the magnitude of [Ca2+]i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP3) generation, the Mn2+ influx, and the capacity of mitochondrial Ca2+ accumulation. Furthermore, the sensitivity of IP3 in the Ca2+ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca2+]i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP3, and the slowed rate of Ca2+ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca2+ signaling cascade in NG108-15 cells.     

The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum

In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.     

Effect of phorbol ester, 12-deoxyphorbol 13-isobutylate (DPB), on muscle tension and cytosolic Ca2+ in rat anococcygeus muscle

Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca2+ ([Ca2+]i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10(-6) M) induced a large contraction and an elevation of [Ca2+]i level in rat aorta and small and rhythmic changes in tension and [Ca2+]i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca2+]i level elevated by high K+, ionomycin or beta-escin in the anococcygeus muscle. 3) In the beta-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca(2+)-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10(-5) M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca2+]i and the DPB-induced enhancement may be due to a Ca2+ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca2+ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca2+]i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.     

Pindolol does not act only on 5-HT1A receptors in augmenting antidepressant activity in the mouse forced swimming test

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pHo) cause increases in Ins(1,4,5)P3 and cytosolic calcium ([Ca2+]c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pHo 7. 4, PCO2 36 mm Hg), metabolic alkalosis (pHo 7.7, PCO2 36 mm Hg), or respiratory alkalosis (pHo 7.7, PCO2 19 mm Hg). Intracellular pH (pHi) and [Ca2+]c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P3 was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pHi from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P3, as we showed previously. Metabolic and respiratory alkalosis increased [Ca2+]c about 80% and 110%, respectively. Neither Ins(1,4,5)P3 nor [Ca2+]c increased in cells treated with aCSF that produced control pHo with increased pHi (7.3). In contrast, when pHo increased (7.7), but pHi was maintained at control (7.2), Ins(1,4,5)P3 increased from 123 pmol/well to 307 pmol/well and [Ca2+]c increased 46%. However, the increase of [Ca2+]c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P3 with resultant elevation in [Ca2+]c. While the Ins(1,4,5)P3 signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.     

Adrenergic signaling and second messenger production in hepatocytes of two fish species

The activation of cAMP and inositol 1,4,5-trisphosphate (IP3) transduction pathways by epinephrine (EPI) has been studied in hepatocytes and hepatic membranes of two teleost species, the American eel (Anguilla rostrata) and the black bullhead (Ictalurus melas). EPI at 10 microM increased both cAMP and IP3 in a dose- and species-dependent manner. The activation of both systems was the greatest in the eel, and the IP3 system was activated at lower EPI concentrations than the cAMP system. The individual transduction pathways were identified by the EPI-induced increase in cAMP being blocked by propranolol (PROP) but not phentolamine (PHT) and the increase in IP3 by PHT but not PROP. alpha1-Adrenoceptors were characterized by the specific binding of [3H]prazosin (PRZ) to purified hepatic membranes: Kd and Bmax values were 3.0 and 2.2 nM and 108 and 178 fmol x mg-1 protein for eel and bullhead, respectively. PRZ binding was displaced by nonradioactive PRZ and PHT but not by PROP. [3H]IP3 bound specifically to microsomal membranes from hepatic tissues of both species and this binding could best be explained by a two-site binding model; significant differences in binding characteristics were seen between eel and bullhead membranes. This study demonstrates that EPI may act on hepatocytes isolated from the American eel and black bullhead through both signal transduction pathways, inducing changes in glucose production and Ca2+ mobilization previously reported in these species. The quantitative differences between the two species in aspects of this system may reflect differences at the level of the coupling between receptor and messenger production, or at the degradation of the messenger, aspects of the transduction pathways which may be either true species differences or related to the conditions under which the species were held.     

Ca(2+)-activated K+ channel is present in guinea-pig but lacking in rat hepatocytes

The mechanisms of norepinephrine-induced membrane responses in isolated hepatocytes from guinea-pigs and rats were compared using the suction-pipette, patch-clamp method, and intracellular Ca2+ concentration ([Ca2+]i) was measured using the Ca2+ fluorescent dye, Quin 2. The resting membrane potentials of isolated guinea-pig hepatocytes were -50 +/- 1 mV (mean +/- SD; n = 38), which is similar to that previously reported in rat hepatocytes by Sawanobori et al. (J Cell Physiol 139: 580-585, 1989). In guinea-pig hepatocytes, norepinephrine (6 microM) caused a membrane hyperpolarization, and norepinephrine (6 microM) or Ca(2+)-ionophore (A23187) (0.4 microM) caused a corresponding outward current. The sensitive current produced by norepinephrine and Ca(2+)-ionophore reversed its polarity at -74 +/- 9 mV (n = 7). The single channel recorded by cell-attached patch and inside-out patch had mean conductance of around 20 + 1 pS and was activated by 1 microM [Ca2+]i. On the other hand, neither norepinephrine (6-20 microM) nor Ca(2+)-ionophore (A 23187) (0.4 microM) caused any change in membrane potential and current in rat hepatocytes, whereas norepinephrine increased [Ca2+]i both in rat and guinea-pig hepatocytes to a similar degree. In the single-channel recording, we recorded single channels that had a mean conductance of 109.8 +/- 17.7 pS different from around 20 pS in guinea-pig. In inside-out patches, increased Ca2+ concentration from 10(-6) to 10(-3) M at the intracellular face of the membrane did not modify the single channel of rat hepatocytes. These results indicate that increased [Ca2+]i activates this channel in guinea-pigs, but that the channel activated by increased [Ca2+]i is lacking in rat hepatocytes membrane. Therefore, different mechanism operates in different species of liver cells to keep the constant state.     

Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca2+ and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst5 receptors (CHOsst5 cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC50 7.10) and UTP (pEC50) 5.14) caused concentration-dependent increases in [Ca2+]i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca2+]i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P3 but the SRIF maximum was about 30% of that to UTP. Removal of [Ca2+]e attenuated the SRIF-induced peak rise in [Ca2+]i but had no effect on the peak increases in Ins(1,4,5)P3. UTP-induced increases in [Ca2+]i and Ins(1,4,5)P3 were attenuated in the absence of [Ca2+]e. Following pre-exposure to SRIF (1 microM) or UTP (100 microM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca2+]e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca2+]e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca2+]i in CHOsst5 cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca2+ and release from an Ins(1,4,5)P3 sensitive Ca2+ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca2+]i could be demonstrated in the presence or absence of extracellular Ca2+ respectively, and the latter appeared to involve depletion of a common intracellular Ca2+ store.     

The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol 1,4,5-trisphosphate production

Rotavirus infection is the leading cause of severe diarrhea in infants and young children worldwide. The rotavirus nonstructural protein NSP4 acts as a viral enterotoxin to induce diarrhea and causes Ca2+-dependent transepithelial Cl- secretion in young mice. The cellular basis of this phenomenon was investigated in an in vitro cell line model for the human intestine. Intracellular calcium concentration ([Ca2+]i) was monitored in fura-2-loaded HT-29 cells using microscope-based fluorescence imaging. NSP4 (1 nM to 5 microM) induced both Ca2+ release from intracellular stores and plasmalemma Ca2+ influx. During NSP4-induced [Ca2+]i mobilization, [Na+]i homeostasis was not disrupted, demonstrating that NSP4 selectively regulated extracellular Ca2+ entry into these cells. The ED50 of the NSP4 effect on peak [Ca2+]i mobilization was 4.6 +/- 0.8 nM. Pretreatment of cells with either 2.3 x 10(-3) units/ml trypsin or 4.4 x 10(-2) units/ml chymotrypsin for 1-10 min abolished the NSP4-induced [Ca2+]i mobilization. Superfusing cells with U-73122, an inhibitor of phospholipase C, ablated the NSP4 response. NSP4 induced a rapid onset and transient stimulation of inositol 1,4,5-trisphosphate (IP3) production in an IP3-specific radioreceptor assay. Taken together, these results suggest that NSP4 mobilizes [Ca2+]i in human intestinal cells through receptor-mediated phospholipase C activation and IP3 production.     

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arginine vasopressin triggers intracellular calcium release, a calcium-activated potassium current and exocytosis in identified rat corticotropes

Arginine vasopressin (AVP) stimulates the secretion of ACTH from pituitary corticotropes. We investigated the action of AVP in single corticotropes of male rats. Corticotropes were identified with the reverse hemolytic plaque assay using antibodies against ACTH. Using the whole-cell recording technique in conjunction with the fluorescent Ca2+ indicator, indo-1 to measure the concentration of cytosolic free Ca2+ ([Ca2+]i), we show that AVP triggers a transient and plateau pattern of Ca2+ signal. The [Ca2+]i elevation activates the apamin-sensitive Ca2+-activated K+ current, which, in turn, causes membrane hyperpolarization. The Ca2+ signal can be elicited in the absence of extracellular Ca2+ and is mimicked by intracellular inositol 1,4,5-trisphosphate (IP3). Both GDP-beta-S and heparin inhibit the AVP response. Thus, AVP triggers intracellular Ca2+ release from the (IP3)-sensitive store via a GTP binding protein-coupled phosphoinositide pathway. Using the high temporal resolution capacitance measurement to detect exocytosis in single corticotropes, we show that a burst of exocytosis is evoked during the AVP-triggered [Ca2+]i elevation. Exocytosis can also be triggered when Ca2+ is released directly from the IP3-sensitive store via flash photolysis of caged IP3. We conclude that AVP-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release from the IP3-sensitive store.     

Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction

To characterize the calcium (Ca2+)-releasing effects of histamine and GTP gamma S, the drug-induced tension developments were measured in beta-escin-treated skinned longitudinal smooth muscle of guinea pig ileum. Intracellular Ca2+ stores were loaded with Ca2+ by incubating the muscle for 10 min in a Ca(2+)-containing solution. Histamine (10-100 microM), applied after Ca(2+)-loading, produced a transient rise in tension. The effect of histamine was not preserved after treatment with 20 mM caffeine, a Ca(2+)-store releaser. The effect of histamine was potentiated by GTP; inhibited by GDP beta S, an antagonist of GTP for binding to G-proteins; or heparin, an antagonist of inositol 1,4,5-trisphosphate (IP3) for binding to its receptor; and mimicked by IP3. When GTP gamma S (20 microM) was applied and continued to be present for 15 min, a transient rise in tension followed by a small, sustained rise in tension was elicited. The effect of GTP gamma S was completely inhibited by GDP beta S. The initial, transient component of the biphasic GTP gamma S response was abolished or markedly inhibited after treatment with caffeine, heparin or the calcium ionophore A23187. The present results suggest that histamine and GTP gamma S cause a release of Ca2+ from caffeine-sensitive stores which is mediated by IP3 formed through a G-protein-coupled mechanism. The GTP gamma S-induced Ca2+ release is not considered to involve such an IP3-independent process as described in chemically-skinned arterial muscle.     

New developments in the molecular pharmacology of the myo-inositol 1,4,5-trisphosphate receptor

Receptor-mediated activation of phospholipase C to generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a ubiquitous signalling pathway in mammalian systems. A family of three IP3 receptor subtype monomers form functional tetramers, which act as effectors for Ins(1,4,5)P3, providing a ligand-gated channel that allows Ca2+ ions to move between cellular compartments. As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, Ins(1,4,5)P3 is considered to be a second messenger that mobilizes Ca2+ from intracellular stores. Ca2+ store mobilization by Ins(1,4,5)P3 can be shown to contribute to a variety of physiological and pathophysiological phenomena, and therefore the IP3 receptor represents a novel, potential pharmacological target. In this article, Rob Wilcox and colleagues review recent developments in IP3 receptor pharmacology, with particular emphasis on ligand molecular recognition by this receptor-channel complex. The potential for designing non-inositol phosphate-based agonists and antagonists is also discussed.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

Sensing and refilling calcium stores in an excitable cell

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.