Involvement of calcium channels in short-term desensitization of muscarinic receptor-mediated cyclic GMP formation in mouse neuroblastoma cells.

Incubation of mouse neuroblastoma cells (clone N1E-115) with Mn2+ resulted in a rapid and transient increase in cyclic GMP formation. This effect appears to be due to an increase in calcium influx because it did not occur in the absence of extracellular calcium or in the presence of verapamil, a calcium transport inhibitor. In addition, Mn2+ inhibited muscarinic receptor-mediated cyclic GMP responses. The ability of Mn2+ to increase cyclic GMP levels was markedly diminished in cells desensitized to the effects of carbamoylcholine, suggesting that this densensitization involves inactivation of calcium entry.     

Measurement of cytosolic free calcium concentration in relation to insulin release in normal rat pancreatic islets

The effects of glucose (16.7 mM), potassium (50 mM), forskolin (20 microM), dibutyryl cyclic AMP (1 mM) and arachidonic acid (82 microM) upon insulin release and cytosolic free calcium concentrations ([Ca2+]i) were investigated in normal rat pancreatic islets. Potassium, forskolin and dibutyryl cyclic AMP significantly raised [Ca2+]i, but elicited only minimal insulin release. In the absence of extracellular Ca2+, glucose evoked insulin release, but failed to augment [Ca2+]i. Arachidonic acid increased [Ca2+]i both in Ca-depleted and -repleted medium, but promoted insulin release only in Ca2+-repleted environment. These new observations clearly demonstrate by direct measurements that although [Ca2+]i is an important factor in exocytotic insulin release, its effect is subject to amplification or antagonism.     

Distinct regulations by calcium of cyclic GMP levels and catecholamine secretion in isolated bovine adrenal chromaffin cells

The effects of various calcium-dependent secretagogues on cyclic GMP levels and catecholamine (CA) secretion were measured in a preparation of bovine adrenal chromaffin cells. The secretory effect of acetylcholine (ACh; 8--10 fold stimulation) was mimicked by nicotine but not muscarine. Three--five fold stimulations of cyclic GMP levels were also obtained with ACh and muscarine but not nicotine. High concentration of K+, and the ionophore A23187, also elevated cyclic GMP levels. However, secretion produced by veratridine, ouabain, and the ionophore X537A was not accompanied by any rise in cyclic GMP levels. Removal of extracellular calcium significantly decreased both basal levels of CA secretion and of cyclic GMP and completely abolished their stimulation by ACh. The half-maximal effects of calcium on the cholinergic stimulations of cyclic GMP levels and of CA secretion were observed at 0.2 and 2.5 mM, respectively. Substitution of Ca2+ by Sr2+ was more effective in maintaining the cyclic GMP response than the secretory response. The calcium channel blockers Co2+, Mg2+ and Ni2+ inhibited the cholinergic stimulation of cyclic GMP more than that of CA release. On the other hand, the organic calcium channel blockers, verapamil and methoxyverapamil (D--600) were more effective antagonists of the secretory response. These data indicate that the cholinergic stimulations of CA secretion and of cyclic GMP levels in bovine adrenal chromaffin cells are regulated by calcium via two distinct mechanisms.     

Effects of prostaglandin F2 alpha and other potential secretagogues on oxytocin secretion and second messenger metabolism in the ovine corpus luteum in vitro

Release of oxytocin by sliced or minced sheep luteal tissue in vitro was stimulated up to 1.6- and 2.3-fold by arachidonic acid and the calcium ionophore A23187 respectively. Prostaglandin (PG) F2 alpha and the PGF2 alpha analogue cloprostenol, and other potential agonists known to be active in vivo, including noradrenaline and acetylcholine, were ineffective, as was the phorbol ester tetradecanoylphorbol acetate (TPA). The ineffectiveness of PGF2 alpha was not due to a general unresponsiveness of the tissue in vitro, as PGF2 alpha reduced LH stimulation of tissue concentrations of cyclic AMP and activated inositol lipid hydrolysis. The effect of arachidonic acid was accompanied by release from the tissue of the cytosolic enzyme lactate dehydrogenase (at arachidonic acid concentrations below those required to release oxytocin) and its effect on oxytocin and lactate dehydrogenase release was mimicked by oleic and linolenic acids; arachidonic acid was concluded to act by a non-physiological physicochemical effect without conversion to an eicosanoid. As PGF2 alpha in vitro is known to raise intracellular Ca2+ concentrations in the large luteal cells that secrete oxytocin, and as A23187 stimulates oxytocin release in vitro in the presence and absence of TPA, it is concluded that in-vitro incubation results in an artifactual blockade of the oxytocin-releasing action of PGF2 alpha at an unidentified point distal to the effect on intracellular Ca2+.     

Bradykinin stimulates phospholipid methylation, calcium influx, prostaglandin formation, and cAMP accumulation in human fibroblasts.

The biochemical events that lead to bradykinin stimulation of cAMP accumulation in human fibroblasts were examined. Treatment of human fibroblasts with bradykinin increases phospholipid methylation, Ca2+ influx, arachidonic acid release, prostaglandin formation, and cAMP content. The dose-response curves of bradykinin for the increase in the above changes were similar. In human fibroblasts, exogenous arachidonic acid was mainly incorporated into phosphatidylcholine, followed by phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. Bradykinin caused a release of arachidonic acid from methylated phospholipids (phosphatidylcholine) and phosphatidylinositol. 3-Deazaadenosine, a methyltransferase inhibitor, almost completely inhibited bradykinin-stimulated phospholipid methylation and Ca2+ influx and partially reduced arachidonic acid release and prostaglandin formation but had no effect on cAMP formation. Mepacrine, a phospholipase inhibitor, blocked bradykinin-induced arachidonic acid release, prostaglandin release, and cAMP accumulation. Indomethacin, a cyclooxygenase inhibitor, blocked the effect of bradykinin on cAMP accumulation. Prostaglandins E1 and E2, but not F2 alpha, increased accumulation of cAMP. These observations indicate that bradykinin generates cAMP via arachidonic acid release and subsequent formation of prostaglandins. Our findings suggest that arachidonic acid can arise from either phosphatidylcholine synthesized by the methylation pathway or phosphatidylinositol.     

Distribution of nitric oxide synthase and secretory role of exogenous nitric oxide in the isolated rat pancreas.

BACKGROUND: Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. METHODS: NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method. Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca2+]i) was measured in dispersed pancreatic acinar cells. RESULTS: NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca2+]i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca2+]i. CONCLUSION: NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nerve terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.     

Stimulation of arachidonic acid release and inhibition of mitogenesis by cloned genes for muscarinic receptor subtypes stably expressed in A9 L cells.

A family of genes encoding four distinct muscarinic receptors (designated m1-m4) has been cloned and stably expressed in A9 L cells. When the m1 and m3 receptors were stimulated with carbachol, there was a rapid rise of liberated arachidonic acid, inositol phosphates, and cAMP, while m2 and m4 receptor stimulation had no detectable stimulation of these second messengers. Pretreatment with phorbol 12-myristate 13-acetate (PMA) caused a marked acceleration and amplification of m1 and m3 receptor-mediated arachidonic acid release. In contrast, m1- and m3-mediated inositol phosphate formation was inhibited by the same PMA pretreatment. Arachidonic acid release was unaffected by manipulations of cAMP levels. Arachidonic acid production was inhibited by calcium-free medium and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; an inhibitor of cytosolic calcium mobilization) yet was unaffected by verapamil, a calcium-channel blocker. These experiments show that arachidonic acid release induced by the m1 and m3 receptors is regulated independently of phospholipase C and cAMP accumulation. Carbachol stimulation of the m1 and m3 cAMP accumulation. Carbachol stimulation of the m1 and m3 receptors also markedly decreased mitogenesis as measured by thymidine incorporation. The m1 receptor-mediated inhibition of mitogenesis could be partially blocked by indomethacin, a cyclooxygenase inhibitor. The inhibition of mitogenesis could be mimicked by cAMP elevation.     

Inhibition of the acetylcholine-induced secretion of bovine growth hormone by somatostatin.

The effects of calcium deprivation, somatostatin and verapamil on the stimulation of growth-hormone release and the alteration of pituitary metabolism in response to acetylcholine were investigated. Calcium deprivation decreased the rises in growth-hormone secretion and in cyclic GMP content in response to 25 μM acetylcholine but did not prevent the increased incorporation of ³²P into phosphatidyl inositol. Somatostatin (1 μg/ml) prevented the rise in growth-hormone secretion and inhibited the efflux of ⁴⁵Ca in response to acetylcholine (25 μM) but did not modify the increase in cyclic GMP content or phosphatidyl inositol labelling. Verapamil (50 μM) did not affect any of the responses to acetylcholine (25 μM). The data suggest that acetylcholine stimulates calcium mobilization from tissue stores and that somatostatin can prevent this mobilization. The relevance of this to the inhibition of secretion by somatostatin is discussed.     

The importance of calcium ions for the regulation of guanosine 3':5'-cyclic monophosphage levels

Guanosine 3':5'-cyclic monosphosphate (cyclic GMP) levels in the ductus deferens of the rat were increased 2- to 3-fold by acetylcholine (10-1000 muM) or by 125 mM KCl, while adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were not changed. After incubation for 30 min in the absence of Ca(++), cyclic GMP control levels were decreased by 85% and were not affected by acetylcholine or KCl. The readdition of Ca(++) (1.8 mM) for 3 min to Ca(++)-deprived tissue partially restored basal cyclic GMP levels and the effects of acetylcholine and KCl. The addition of Sr(++) (3.6 mM) or of Ba(++) (1.8 or 10 mM) also caused an increase in basal cyclic GMP in Ca(++)-deprived tissue. Cyclic AMP levels were not significantly changed under any of these conditions. The addition of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.1 mM), to ductus deferentes increased the amount of cyclic AMP about 50% and that of cyclic GMP about 2-fold. The later effect also depended on the presence of Ca(++). 1-Methyl-3-isobutylxanthine (0.1 mM) increased cyclic GMP and cyclic AMP levels in slices of rat submaxillary glands. Methacholine increased cyclic GMP if added in the presence of methyl isobutylxanthine. Cyclic GMP control levels and the effect of methyl isobutylxanthine were unchanged by Ca(++) omission, but the effect of methacholine was abolished.These findings indicate that calcium ions are important for the control of cyclic GMP levels in these tissues.     

The effect of linoleic and arachidonic acid derivatives on calcium transport in vesicles from cardiac sarcoplasmic reticulum

The effect of linoleic and arachidonic acid derivatives on ATP-dependent calcium transport was studied in the isolated vesicles from cardiac sarcoplasmic reticulum of guinea-pigs. Oxidation products of linoleic and arachidonic acids, obtained either by autoxidation or incubation with soybean lipoxygenase, effectively blocked in a dose-dependent manner, the net influx of calcium in the absence or presence of 5 mM of oxalate. Unoxidized fatty acids were much weaker at lower concentrations as compared to their oxidized counterparts, except the lipoxygenase-generated product of arachidonic acid which had only a marginal effect even at high concentrations. Autoxidation products of arachidonic acid were the most potent inhibitors of calcium transport. Likewise, autoxidation products of linoleic and arachidonic acids and lipoxygenase-generated products of linoleic acid induced a dose-dependent release of calcium from vesicles previously loaded with 45Ca, and release was further enhanced in the presence of 0.5 mM of EGTA. In contrast, lipoxygenase metabolites of arachidonic acid caused a transient increase in net calcium content. The effect of the fatty acid derivatives on calcium transport did not appear to be due either to the inhibition of Ca2+-ATPase activity or to a non-specific detergent-like action. The effects of oxidized fatty acids, on ATP-dependent calcium accumulation into and release from cardiac microsomal fraction were similar but less potent than those of classical calcium ionophores, X537A or A23187.     

Involvement of MAP kinases in the control of cPLA(2) and arachidonic acid release in endothelial cells

Cytosolic Phospholipase A(2) (cPLA(2)) has been implicated in receptor-mediated release of arachidonic acid from membrane phospholipids, the limiting step in prostacyclin and other eicosanoid production. Its activity is controlled by Ca(++) levels and enzymatically regulated phosphorylation. The purpose of this study was to assess the importance of phosphorylation of cPLA(2) in human umbilical vein endothelial cells and to identify the kinases involved. Inhibitors were used to study the pathways leading to phosphorylation and activation of mitogen activated protein kinases (MAP-kinases) and cPLA(2), as well as release of arachidonic acid and prostacyclin production after stimulation with different agonists. We have found that agonists that release arachidonic acid, including histamine, thrombin, AlF(4)(-), and pervanadate, all activate the MAP kinases ERK, p38 and JNK and cause phosphorylation of cPLA(2). Agonist specific differences in the signal transduction pathways included variable contribution of tyrosine phosphorylation, protein kinase C and ERK activity, and different effects of pertussis toxin. Treatment with PD98059 (inhibitor of ERK-activation) or SB203580 (inhibitor of p38) caused partial decrease in arachidonic acid release and cPLA(2) activity. In contrast the nonspecific protein kinase inhibitor staurosporin completely inhibited cPLA(2) activity. We conclude that in endothelial cells arachidonic acid release is largely mediated by cPLA(2) through agonist-specific pathways. The MAP kinases ERK and p38 both have demonstrable but not major effect on agonist stimulated arachidonic acid release and the data suggest that an additional unidentified kinase also has a role.     

Further Studies on the Effects of the Intracellular Histamine Antagonist DPPE on Platelet Function

SUMMARY. The role of intracellular histamine in the activation of human platelets was explored using the novel histamine antagonist N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE). DPPE inhibited aggregation, [(32)P]-phosphatidic acid production (an index of phospholipase C activity) and the increase in cytosolic calcium ([Ca(2+)]i) in response to collagen. In contrast, while at higher concentrations DPPE inhibited aggregation in response to ADP and the thromboxane mimetic EP171, it failed to inhibit EP171-induced [(32)P]-phosphatidic acid production or increases in [Ca(2+)]i elicted by either ADP or EP171. Collagen but neither ADP nor EP171 caused the significant formation of intracellular histamine. The biochemical changes induced by collagen have been shown previously to be, at least partly, secondary to thromboxane generation. Collagen-induced arachidonic acid release was therefore assessed. DPPE inhibited the release of arachidonic acid in response to collagen but had no effect on its subsequent conversion to thromboxane. The findings imply that intracellular histamine acts at an early stage in collagen-induced platelet activation, most likely prior to arachidonic acid release, and further that DPPE inhibits ADP and EP171-induced activation by a non-histamine related effect.     

Regulation of coronary artery tone in relation to the activation of signal transductors that regulate calcium homeostasis

Muscle tone of coronary arteries is regulated by free calcium concentration in the myoplasm. Various agonists, autacoids and putative peptides modify the calcium concentration directly or through actions of second messengers (signal transductors), such as cyclic guanosine monophosphate (GMP), cyclic adenosine monophosphate (AMP), inositol 1,4,5-trisphosphate, diacylglycerol or calmodulin. For example, acetylcholine (in the presence of intact endothelium cells), alpha-human natriuretic peptide or nitrate compounds increase the amount of cyclic GMP and isoproterenol, prostacyclin (prostaglandin I2) or vasoactive intestinal polypeptide increases cyclic AMP. Both cyclic nucleotides reduce free calcium concentration. On the other hand, acetylcholine (in the presence or absence of endothelium cells), norepinephrine or thromboxane A2 increases inositol 1,4,5-trisphosphate and diacylglycerol, thus causing the increase in the free calcium concentration, whereas vasoactive intestinal peptide and alpha-human natriuretic peptide reduce them. Calmodulin acts as an internal calcium receptor for regulation of the contractile machinary. Regulation of calcium homeostasis in relation to the muscle tone in the coronary arteries including other vascular tissues is discussed together with the role of second messengers.     

Regulation of arachidonic acid release by calcium influx in human endothelial cells.

In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca2+ pumps, time- and dose-dependently increased the release of [1-14C]-arachidonic acid. This release was inhibited by AACOCF3, a selective inhibitor of cytosolic phospholipase A2 (PLA2). In the absence of Ca2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca2+ concentration ([Ca2+]i). In the presence of Ca2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca2+ buffer, or with SK&F 96365, a receptor-operated Ca2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca2+ response to stimulation: Ca2+ peak and plateau phase, reflecting Ca2+ mobilization from internal stores and Ca2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca2+]i values measured at the influx phase with a 80 nM [Ca2+]i threshold, similar to that reported for PLA2 translocation. This suggests that Ca2+ entry from the extracellular space is essential for activating cytosolic PLA2 in human endothelial cells.     

Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle

The purpose of this study was to investigate the effects of the intracellular messenger cyclic GMP (cGMP) on sequestration of cytosolic calcium (Ca2+) into the intracellular Ca2+ store (the sarcoplasmic reticulum) of vascular smooth muscle. Using saponin-skinned primary cultures of rat aortic smooth muscle, we investigated the effect of cGMP on 45Ca uptake in monolayers of cells. The intracellular store was loaded with Ca2+ by exposing the skinned cells to a 45Ca-labeled 1-microM free Ca2+-containing solution for varying durations (0-20 minutes). Addition of 10 microM cGMP to six monolayers increased both the initial Ca2+ uptake at 2 minutes (control, 240 +/- 8 pmol Ca2+/10(6) cells; + cGMP 295 +/- 7; mean +/- SEM; n = 6, p less than 0.01) and the final steady-state uptake reached at 20 minutes (control, 0.96 +/- 0.03 nmol Ca2+/10(6) cells; + cGMP 1.12 +/- 0.03, p less than 0.02). This stimulation of uptake was quantitatively similar to that caused by 10 microM cyclic AMP. It occurred at varying ambient cytosolic Ca2+ concentrations (0.1-1.0 microM Ca2+) and was not further enhanced by addition of 10 microM cGMP-dependent protein kinase. The dose-response of stimulation of Ca2+ uptake with cGMP indicated an ED50 of 5 nM cGMP. The release of Ca2+ from the sarcoplasmic reticulum in response to inositol 1,4,5-trisphosphate or caffeine was unaffected by cGMP. We conclude that the relaxation of vascular smooth muscle with cGMP-producing vasodilators is mediated in part by sequestration of cytosolic Ca2+ by the sarcoplasmic reticulum.     

Arachidonic acid-induced LH release is ATP-independent and insensitive to N-ethyl maleimide.

The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal alpha-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In alpha-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis.     

Mediation of flow-dependent arterial dilation by endothelial cells

When blood flow in a large artery is increased the vessel dilates. This flow-dependent dilation requires endothelial cells, and is not mediated by an ascending message from the microcirculation or a myogenic mechanism. Adrenergic, cholinergic, or ganglionic blockade does not alter the dilation response. Inhibition of cyclo-oxygenase by indomethacin has no effect, but inhibition of both lipoxygenase and cyclo-oxygenase by 5,8,11,14-eicosatetraynoic acid (ETYA) inhibits the dilation and shifts the acetylcholine dose response curve to the right. Inhibition of guanylate cyclase by methylene blue blocks the dilation response and shifts the acetylcholine dose response curve to the right. This suggests that both cyclic GMP and a nonprostaglandin metabolite of arachidonic acid are involved in the dilation response to increased flow. We propose that increased blood flow initiates an initial response, which results in endothelial cell production and release of a nonprostaglandin metabolite of arachidonic acid. This metabolite stimulates vascular smooth muscle guanylate cyclase, leading to increased cyclic GMP and vasodilation.     

Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells.

In FRTL5 rat thyroid cells, norepinephrine, by interacting with alpha 1-adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple alpha 1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized FRTL5 cells, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]), which activates many GTP-binding proteins, stimulated inositol phosphate formation and arachidonic acid release. Neomycin inhibited GTP[gamma-S]-stimulated inositol phosphate formation but was without effect on GTP[gamma-S]-stimulated arachidonic acid release, suggesting that separate GTP-binding proteins mediate each process. In addition, pertussis toxin inhibited norepinephrine-stimulated arachidonic acid release but not norepinephrine-stimulated inositol phosphate formation. Norepinephrine-stimulated arachidonic acid release but not inositol phosphate formation was also inhibited by decreased extracellular calcium and by TMB-8, suggesting a role for a phospholipase A2. To confirm that arachidonic acid was released by a phospholipase A2, FRTL5 membranes were incubated with 1-acyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine. GTP[gamma-S] slightly stimulated arachidonic acid release, whereas norepinephrine acted synergistically with GTP[gamma-S] to stimulate arachidonic acid release. The results show that phospholipase C and phospholipase A2 are activated by alpha 1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-insensitive.     

Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.     

Effects of carbamylcholine and ionophore A-23187 on cyclic 3',5'-AMP and cyclic 3',5'-GMP accumulation in dog-thyroid slices.

Carbamylcholine and acetylcholine through a muscarinic type of receptor, KCl, ionophore A-23187 and NaF increased cyclic GMP accumulation in dog-thyroid slices. These effects were abolished in calcium-depleted slices, which findings confirm that Ca2+ is required for cyclic GMP accumulation. All these agents depressed the accumulation of cyclic AMP in TSH-stimulated slices. KCl and NaF depressed cyclic AMP accumulation in TSH-treated slices even when they had been depleted of Ca2+. This suggests a cyclic GMP- and Ca2+-independent mechanism. The absence of inhibition of the effects of the ionophore, NaF and KCl in the presence of atropine suggests that these drugs do not act by inducing the release of acetylcholine in the slices. The effects of carbamylcholine and ionophore A-23187 on cyclic GMP accumulation and protein iodination were reversible; the inhibitions of TSH-induced cyclic AMP accumulation and secretion were non-competitive and were not accompanied by a depression of ATP levels. All these effects were greatly decreased in the absence of extracellular Ca2+. These data suggest that carbamylcholine and ionophore A-23187 act mainly by increasing the influx of extracellular Ca2+ in thyroid cells. However, the persistence of some carbamylcholine effect in the absence of Ca2+ in the medium suggests that this agent may also trigger the release of Ca2+ from an intrafollicular pool. The kinetics of action of carbamylcholine are compatible with a role of cyclic GMP in the inhibition of cyclic AMP accumulation. However, with the ionophore, the depression of cyclic AMP accumulation was much longer than the rise of cyclic GMP, which suggests a mechanism independent of cyclic GMP.