[Consequences of glaucoma on circadian and central visual systems]. / Impact de la pathologie glaucomateuse sur les voies visuelles centrales et les structures circadiennes.

Glaucoma is a chronic optic neuropathy leading to a degeneration of retinal ganglion cells. There is accumulating evidence that glaucomatous damage extends from retinal ganglion cells to vision centers in the brain. Degenerative changes are observed in magnocellular, parvocellular, and koniocellular pathways in the lateral geniculate nucleus, and these changes are related to intraocular pressure and the severity of optic nerve damage. In addition, recent studies show that there are also changes in the visual cortex in relation to varying degrees of retinal ganglion cell loss. In a rat model of glaucoma, we have recently demonstrated a reduction of retinal projections of retinal ganglion cells, not only on the visual system but also on the suprachiasmatic nucleus. Human studies suggest that the ganglion cell degeneration caused by glaucoma could lead to a lesion of the retinohypothalamic tract, which permits the synchronization of circadian rhythms.

Neuroprotection of retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular hypertension.

PURPOSE: To examine the neuroprotective effect of the alpha(2)-adrenergic agonist brimonidine in a chronic ocular hypertension model. METHODS: Intraocular pressure (IOP) was elevated by laser photocoagulation of episcleral and limbal veins. Retinal ganglion cell loss was evaluated in wholemounted retinas. Brimonidine or timolol was administered, either at the time of or 10 days after IOP elevation and continued for 3 weeks. Drug-related immunohistochemical changes in glial fibrillary acidic protein (GFAP) were also determined after 3 weeks. RESULTS: Laser treatment caused a twofold IOP increase over baseline that was maintained for 2 months. A time-dependent loss of ganglion cells occurred with elevated IOP. Systemic administration of brimonidine or timolol caused little decrease in IOP. After 3 weeks of elevated IOP, ganglion cell loss in control rats was 33% +/- 3%. Brimonidine reduced the progressive loss of ganglion cells to 26% +/- 1% and 15% +/- 2% at doses of 0.5 and 1 mg/kg. d, respectively. Timolol had no effect. Ten days of high IOP resulted in 22% +/- 4% ganglion cell loss. Brimonidine administration initiated 10 days after IOP elevation prevented any further loss of ganglion cells. In vehicle- or timolol-treated rats, ganglion cell loss continued to 33%. The increase in immunoreactivity of GFAP in ocular hypertensive retinas was attenuated by brimonidine. CONCLUSIONS: Systemic application of brimonidine or timolol had little effect on IOP. Brimonidine, but not timolol, showed significant protection of retinal ganglion cells when applied at the time of IOP elevation and prevented further cell loss when applied after IOP was elevated. This indicates that brimonidine has a neuroprotective activity unrelated to its effect on ocular hypotension.

Alpha-2 adrenergic receptor agonists are neuroprotective in experimental models of glaucoma.

PURPOSE: The glaucomas are characterized by chronic progressive ganglion cell loss over many years. A drug with neuroprotective activity should increase the resistance of retinal ganglion cells (RGC) to chronic stress or injury and therefore enhance survival. Brimonidine is a highly selective and potent alpha-2 adrenergic receptor agonist, which lowers intra-ocular pressure (IOP) and is neuroprotective. Immunohistochemistry data have shown that the specific receptor targets, the alpha-2 receptors, are located in the inner retina. METHODS: Brimonidine 0.1 mg/kg given intraperitoneally promoted RGC survival compared with vehicle using the optic nerve crush model even when administered up to 24 hours before injury. Using the chronic ocular hypertensive rat model, brimonidine 1 mg/kg/day (with osmotic pump) significantly prevented the loss of RGCs when compared with vehicle or timolol. This ability was due to the neuroprotective action of brimonidine, since it did not affect IOP. In addition, brimonidine 0.1 mg/day reached concentrations in the retina of Sprague-Dawley rats within 30 minutes of injection, which was sufficient to activate the alpha-2 receptor (> or = 2 nM) and maintained these concentrations for 6 hours. CONCLUSIONS: Having demonstrated that: a. the specific receptor target of brimonidine is located in the retina, which is important for optic neuroprotection, b. the agent shows neuroprotective ability in animal models, c. pharmacological concentrations of the drug can be reached in the retina, clinical trial has been initiated to determine whether brimonidine is neuroprotective in patients with nonarteritic ischaemic optic neuropathy.

Exploring the potential for subtype-selective muscarinic agonists in glaucoma.

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.

Efficacy and safety of memantine, an NMDA-type open-channel blocker, for reduction of retinal injury associated with experimental glaucoma in rat and monkey.

Glutamatergic excitotoxicity has been implicated as a mechanism for injury in a variety of central nervous system pathologies, including glaucoma. Memantine, an NMDA-type glutamatergic open-channel blocker, has pharmacologic properties that make its efficacy greater under excitotoxic conditions, but lesser under normal conditions. Daily oral dosing for approximately 15 months with 4.0 mg/kg memantine in monkeys yielded plasma concentrations similar to those found in patients who received memantine treatment for Parkinson's disease. This same dose of memantine was not associated with any evidence of an effect on the normal function of the retina and central visual pathways, as indicated by measures of the electroretinogram (ERG) and visually-evoked cortical potential (VECP). Amplitude of the VECP response was reduced in eyes with experimentally induced glaucoma. When compared to vehicle-treated control animals, memantine-treated glaucoma eyes suffered significantly less reduction of VECP amplitude. Preliminary results in a rat model for experimental glaucoma also show that, when compared to control animals, systemic treatment with memantine (10 mg/kg/day) was associated with a significant reduction in glaucoma-induced loss of retinal ganglion cells.

Role of alpha-2 adrenergic receptors in neuroprotection and glaucoma.

The loss of retinal ganglion cells (RGCs) in glaucoma occurs progressively over many years. A neuroprotective drug should enhance survival of RGCs in the presence of chronic stress/injury. Four criteria are proposed for assessing the likely therapeutic utility in human glaucoma of drugs that have demonstrated neuroprotective activity in animal models: 1) A specific receptor target must be in the retina/optic nerve; 2) Activation of the target must trigger pathways that enhance a neuron's resistance to stress/injury and/or suppresses toxic insults; 3) The drug must reach the retina/vitreous at pharmacologic doses; and 4) The neuroprotective activity should be demonstrated in clinical trials. Data are presented that illustrate how the specific and potent alpha-2 agonist, brimonidine, meets these criteria. The alpha-2A receptor was localized in the inner rat retina by immunohistochemistry. Brimonidine reduced the rate of RGC loss in the calibrated rat optic nerve injury model even when dosed 12 and 24 hours before injury, consistent with a long-term enhancement of RGC resistance to stress. Brimonidine was also neuroprotective in the lasered chronic hypertensive rat model, reducing RGC loss over three weeks from 33% to 15%. A clinical trial has been initiated to determine brimonidine's neuroprotective activity in patients with non-arteritic ischemic optic neuropathy.

Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: implications for glaucoma.

Our group recently demonstrated that autoimmune T cells directed against central nervous system-associated myelin antigens protect neurons from secondary degeneration. We further showed that the synthetic peptide copolymer 1 (Cop-1), known to suppress experimental autoimmune encephalomyelitis, can be safely substituted for the natural myelin antigen in both passive and active immunization for neuroprotection of the injured optic nerve. Here we attempted to determine whether similar immunizations are protective from retinal ganglion cell loss resulting from a direct biochemical insult caused, for example, by glutamate (a major mediator of degeneration in acute and chronic optic nerve insults) and in a rat model of ocular hypertension. Passive immunization with T cells reactive to myelin basic protein or active immunization with myelin oligodendrocyte glycoprotein-derived peptide, although neuroprotective after optic nerve injury, was ineffective against glutamate toxicity in mice and rats. In contrast, the number of surviving retinal ganglion cells per square millimeter in glutamate-injected retinas was significantly larger in mice immunized 10 days previously with Cop-1 emulsified in complete Freund's adjuvant than in mice injected with PBS in the same adjuvant (2,133 +/- 270 and 1,329 +/- 121, respectively, mean +/- SEM; P < 0.02). A similar pattern was observed when mice were immunized on the day of glutamate injection (1,777 +/- 101 compared with 1,414 +/- 36; P < 0.05), but not when they were immunized 48 h later. These findings suggest that protection from glutamate toxicity requires reinforcement of the immune system by antigens that are different from those associated with myelin. The use of Cop-1 apparently circumvents this antigen specificity barrier. In the rat ocular hypertension model, which simulates glaucoma, immunization with Cop-1 significantly reduced the retinal ganglion cell loss from 27.8% +/- 6.8% to 4.3% +/- 1.6%, without affecting the intraocular pressure. This study may point the way to a therapy for glaucoma, a neurodegenerative disease of the optic nerve often associated with increased intraocular pressure, as well as for acute and chronic degenerative disorders in which glutamate is a prominent participant.

Characterization of retinal injury using ERG measures obtained with both conventional and multifocal methods in chronic ocular hypertensive primates.

PURPOSE: To characterize, using both conventional and multifocal electroretinogram (ERG) recordings as well as histologic measures, retinal injury in the chronic ocular hypertensive primate model for experimental glaucoma. METHODS: Ocular hypertension was induced in the right eye of 7 cynomolgous monkeys, Macaca fascicularis, using laser injury to the aqueous outflow tissue at the anterior chamber angle. At 16 months after IOP elevation, ERG recordings were made from both eyes of all animals using both conventional and multifocal methods. After electrophysiological recording, animals were killed and retinal samples were radially sectioned for histologic analysis. RESULTS: Histologic measures showed that ocular hypertensive injury was largely or completely limited to a loss of retinal ganglion cells (RGCs). The degree of RGC loss was similar in central and peripheral retina. Amplitudes of conventional ERG responses were mostly unaffected in eyes having severe loss of RGCs, a finding that is consistent with limited injury to photoreceptors, bipolar cells, and amacrine cells. Peaks in both the first- and second-order multifocal ERG responses were attenuated in ocular hypertensive eyes, and amplitude of these peaks was highly correlated with the density of surviving RGCs. CONCLUSIONS: The results are consistent with a conclusion that both first- and second-order components of the multifocal ERG response from the monkey reflect a significant contribution from activity in RGCs and may provide a useful measure for the clinical diagnosis and management of glaucoma.

From the lab to the clinic: activation of an alpha-2 agonist pathway is neuroprotective in models of retinal and optic nerve injury.

PURPOSE: The selective alpha-2 agonist brimonidine was used as a pharmacological probe to activate alpha 2 receptor-mediated neuroprotective signaling pathways and quantitate the enhancement of retinal ganglion cell survival and function in animals with ischemic retinal and optic nerve injury. METHODS: Two animal models were used to achieve different methods of neuronal insult. The first model involved mechanical injury of the rat optic nerve after treatment with a single intraperitoneal (i.p.) dose of brimonidine or a control vehicle. The second model involving acute retinal ischemic/reperfusion injury was used in a variety of experiments in which rats were treated with either intraperitoneal brimonidine or single dose topical brimonidine at various strengths. In all cases retinal ischemia was induced and maintained followed by reperfusion. In some cases, TUNEL staining was performed on histologic sections of the retinas of rats that had been sacrificed after 24 hours. To examine the activation of neuronal survival pathways at the molecular level, rats were injected with i.p. brimonidine followed by the isolation of mRNAs from whole retinas 24 hours after ischemic injury. RESULTS: Intraperitoneal brimonidine enhanced rat RGC survival and function in the partial crush injury model, and neuroprotection was dose-dependent. Topical application of brimonidine 1 hour before injury was effective in decreasing ischemic retinal injury. Ischemic retinas treated with brimonidine resulted with a large decrease in TUNEL staining. CONCLUSIONS: Treatment with the alpha 2 adrenoreceptor agonist brimonidine was found to confer neuroprotection to retinal ganglion cells in two distinct models of neuronal injury resulting from acute retinal ischemia/reperfusion and calibrated optic nerve compression.

Electrophysiological and histological measures of retinal injury in chronic ocular hypertensive monkeys.

PURPOSE: This study was performed to determine whether components of the standard ERG (electroretinogram), multifocal ERG, and flash VECP (visually-evoked cortical potential) response might provide a sensitive measure of retinal ganglion cell injury in a monkey model for chronic ocular hypertension. METHODS: Argon laser treatment of the aqueous outflow tissue was used to induce chronic elevation of intraocular pressure (IOP) in the right eye of 18 young adult cynomolgous monkeys. At 15 months post- IOP elevation, standard methods were used to record ERG and VECP responses. Multifocal ERG responses were also recorded at this time. Loss of retinal ganglion cells due to ocular hypertensive injury was determined by histological analysis of all retinas. RESULTS: Ocular hypertensive retinal injury was associated with a loss of retinal ganglion cells. There was no histological or electrophysiological evidence for injury to any other retinal cell type. Correlation of electrophysiological response amplitudes with histological measures of retinal ganglion cell loss/survival yielded results which suggest that activity in retinal ganglion cells makes a substantial contribution to components of the 30 Hz flicker ERG, the flash VECP, and both first and second order multifocal ERG responses. Of the electrophysiological measures used in this study, multifocal ERG response amplitude had the greatest sensitivity to retinal ganglion cell loss. CONCLUSIONS: Components of the multifocal ERG provide a sensitive measure of ganglion cell injury in a monkey model of chronic ocular hypertension. These same measures may have utility in the clinical diagnosis and management of glaucoma.

Muscarinic receptor subtypes in human iris-ciliary body measured by immunoprecipitation.

PURPOSE: To determine the relative levels of the five muscarinic receptor subtypes in the anterior segment of the human eye. METHODS: Antisera selective for each of the five muscarinic receptor proteins were incubated with [3H]-QNB bound receptors solubilized from human iris sphincter, ciliary muscle, and ciliary processes. Precipitation of the radiolabeled receptor-antibody complexes and scintillation counting enabled quantitation of the subtypes in the various tissues. Reverse transcription-polymerase chain reaction was performed on the tissues and cultured smooth muscle cells derived from them. RESULTS: Approximately 60% to 75% of the muscarinic receptors in the human iris sphincter and ciliary body are the m3 subtype. Lower levels (5% to 10%) of the m2 and m4 receptors are present in these tissues. The m1 receptor (7%) was detected in the ciliary processes and iris sphincter and the m5 receptor (5%), which is usually found only in the central nervous system, was present in the iris sphincter. CONCLUSIONS: The m3 subtype is the predominant muscarinic receptor in the anterior segment of the human eye. The extensive heterogeneity of muscarinic receptors makes it difficult to predict whether subtype-selective drugs will have an improved efficacy and side-effect profile.

Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor.

Calcium signalling was examined in CHO-k1 cells that stably express the m3 subtype of the muscarinic receptor. The calcium indicator Fura-2 was retained in these cells only in the presence of probenecid (1 mM), suggesting that Fura-2 efflux was mediated by an organic anion transporter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspension caused a rapid transient increase in intracellular calcium [Ca]i followed by a smaller sustained plateau phase. The transient rise in [Ca]i was dose-dependent with a threshold response of 89 +/- 18 nM above baseline with 10 nM CCh and a maximum stimulation of 734 +/- 46 nM with 10 microM CCh. This phase was accompanied by a similar dose-dependent stimulation of total inositol phosphate production and was assumed to be generated by release from intracellular stores of the endoplasmic reticulum (ER). The sustained increase in [Ca]i was generated by entry from the extracellular bath since it was blocked by pretreatment with La3+ (1 microM) and was absent when bath calcium was chelated with EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca]i of approximately 90 nM above baseline was observed with CCh concentrations between 50 nM and 10 microM. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation wavelength of 360 nm was likewise maximally stimulated. At lower CCh concentrations (10-50 nM), it was clear that the activation of Ca entry could not be dissociated from a threshold release of Ca from intracellular stores. The phorbol ester PMA, which uncouples the muscarinic receptor from phospholipase C, reduced the transient rise in [Ca]i by approximately 50% with little or no effect on Ca entry at higher CCh levels (> or = 1 microM). At lower CCh concentrations (< or = 100 nM) however, pretreatment with PMA completely blocked all Ca mobilization and supports the contention that Ca entry is coupled to Ca release from stores or to store depletion. The emptying of inositol trisphosphate-sensitive stores with thapsigargin (10 nM) stimulated Ca entry and also the rate of Mn2+ quenching. Store depletion by incubation in Ca-free media likewise stimulated Mn2+ uptake without a rise in [Ca]i. Our data are therefore consistent with a 'capacitative' coupling model, whereby the activation of the plasma membrane receptor leads to an InsP3-induced change in the degree of filling of the ER Ca pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of muscarinic receptors in cultured human iris sphincter and ciliary smooth muscle cells.

Muscarinic receptors present in cultured human iris sphincter and ciliary smooth muscle cells were characterized by both ligand ([3H]QNB binding) and functional (phosphoinositide hydrolysis) studies. Ligand binding studies showed that [3H]QNB represented a single population of binding sites with KD values of 4.02 x 10(-11) M in the ciliary and 5.6 x 10(-11) M in the iris sphincter cells. In competition studies, the selective antagonist, 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP) was the most potent in displacing [3H]QNB with selectivity of 150-350-fold over pirenzepine (M1) and 450-1700-fold over AF-DX 116 (M2). 4-DAMP recognized one site in the iris sphincter cells (Ki = 0.34 nM) but two sites in the ciliary cells (KH = 0.9 nM and KL = 49 nM). 4-DAMP was also the most potent in inhibiting carbachol-induced hydrolysis of inositol phospholipids (PI) in both cell types. However, the IC50 values for PI hydrolysis were several fold lower than those for [3H]QNB binding. Using these selective antagonists, our data supports the presence of functional muscarinic receptors of M3 subtype in human iris sphincter and ciliary cells. It also shows the presence of a second low affinity site in the ciliary smooth muscle cells that is recognized by 4-DAMP.

Effect of histamine on signal transduction in cultured human trabecular meshwork cells.

Stimulation of cultured human trabecular meshwork cells by histamine caused time and dose related increases in inositol phosphates and intracellular free calcium. The increase in inositol trisphosphate (IP3) was immediate and calcium independent while that of inositol monophosphate (IP1) was gradual and calcium dependent. The rise in intracellular calcium was also rapid and occurred as a result of mobilization from intracellular stores and influx from external medium. Histamine also caused time and concentration related de novo synthesis of inositol phospholipids. Mepyramine but not cimetidine inhibited the action of histamine. These results indicate that histamine, via H1 receptor, evokes an early hydrolysis of inositol phospholipids and increase in intracellular free calcium, signals which may be involved with the function of the trabecular meshwork cells.

Prostaglandin F2 alpha effects on intraocular pressure negatively correlate with FP-receptor stimulation.

According to the current working classification for prostanoid receptors, the prostaglandin F2 alpha-sensitive receptor (FP-receptor) may be identified by comparing the rank order of activity of prostaglandin F2 alpha (PGF2 alpha) and its analogues. In order to further understand the pharmacology of PGF2 alpha-induced ocular hypotension, the intraocular pressure response to PGF2 alpha and selected analogues was compared with their rank order of activity in typical FP-receptor preparations such as contraction of the cat iris sphincter and affinity for corporal luteal membrane binding sites. The rank order of potency for decreasing intraocular pressure was as follows: PGF2 alpha greater than PGF1 alpha greater than 16-phenoxytetranor PGF2 alpha greater than 17-phenyltrinor PGF2 alpha = fluprostenol (inactive). For cat iris sphincter contraction, the rank order of potency appears to be fluprostenol = 17-phenyltrinor PGF2 alpha greater than 16-phenoxytetranor PGF2 alpha = PGF2 alpha greater than PGF1 alpha. The rank order of potency for PGF2 alpha analogues in decreasing intraocular pressure appears to negatively correlate with the rank order for cat iris sphincter contraction and literature values for corporal luteal membrane binding. It is concluded that the ocular hypotensive effect of PGF2 alpha is not mediated by the FP-receptor.

Cyclic carbamate analogues of pilocarpine.

A number of pilocarpine analogues containing the (S)-3-ethyl-4-[(4'-imidazolyl)methyl]-2-oxazolidinone (9) structural feature were synthesized from L-histidine. With 1-benzyl-L-histidine as the key intermediate, a regiospecific synthetic route was developed to the N pi-methyl derivative 8. The regiochemistry of the alkylation of the imidazole nucleus was determined by measuring proton cross-ring coupling constants in the high-field 1H NMR. The effects on muscarinic receptors of these variously alkylated derivatives 6-10 were studied on isolated guinea pig ileum. The derivatives in which the imidazole nitrogen was unsubstituted (9), N tau-methylated (10), and N pi-methylated (8) were cholinergic muscarinic agonists with an increasing order of potency; compounds 6 and 7 were inactive. Analogue 8 with the same substitution pattern as pilocarpine was equipotent with pilocarpine, making these hydrolytically stable carbamate derivatives potentially useful drugs.

The role of arachidonic acid metabolism in IL-3-induced proliferation.

The role of arachidonic acid metabolites as second messengers in the IL-3-induced activation of DA-1 cells was examined. By using inhibitors of either the cyclooxygenase (CO) or lipoxygenase (LPO) pathways, we determined that neither prostaglandins nor leukotrienes were involved in signal transduction, since aspirin, indomethacin, meclofenamic acid, and nordihydroguaiaretic acid (NDGA) failed to inhibit the proliferation response of DA-1 cells to IL-3. Furthermore, two combination CO/LPO inhibitors, benoxaprofen and BW755c, failed to inhibit DA-1 proliferation. A new CO/LPO compound examined, SK&F 86002, did inhibit proliferation (IC50 = 30 microM +/- 14, N = 11), leading us to conclude this drug has other actions besides CO/LPO inhibition. Finally, direct measurement of 3H-arachidonic acid uptake by DA-1 cells failed to show a difference in the amount of 3H-arachidonic acid incorporated in the presence of limiting or saturating amounts of IL-3. We conclude from these data that arachidonic acid metabolites are not involved in transmembrane signalling by IL-3 in DA-1 cells.

Identification of variants of the basophilic leukemia (RBL-2H3) cells that have defective phosphoinositide responses to antigen and stimulants of guanosine 5'-triphosphate-regulatory proteins.

Antigen immunoglobulin E-mediated secretion of histamine from RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and a rise in the concentration of cytosol Ca2+ (calcium signal). Such responses differed among cloned variant lines of the RBL-2H3 cell line from undetectable (1A3 bromodeoxyuridine-resistant (BUDRR), 2B1 BUDRR, and 1B3 BUDRR lines) to about 80% of those in the parent RBL-2H3 cells. In all but one line (1B3 thioguanine-resistant (TgR)), the intensities of the phosphoinositide response and of the calcium signal were correlated with the secretory response. The 1B3 TgR line had no detectable calcium signal (as measured by quin 2 fluorescence or uptake of 45Ca2+) but paradoxically showed modest rates of hydrolysis of inositol phospholipids and of secretion. The responses of the 1B3 TgR line were, however, dependent on the presence of external Ca2+ ions. The induction of secretion with antigen, therefore, was invariably associated with the hydrolysis of inositol phospholipids, but it was not necessarily associated with a change in concentration of cytosol Ca2+. All antigen unresponsive clones could secrete when synergistic signals were induced by exposure to the Ca2+ -ionophore, A23187 and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. These lines, otherwise, had immunoglobulin E receptors and had no obvious defect in their capacity to synthesize the inositol phospholipids or in their phenotypic expression of phospholipase C as measured in cell extracts. One finding of possible relevance to the role of guanosine 5'-triphosphate-regulatory proteins in the activation of phospholipase C was the inability of one antigen-nonresponsive line to respond to NaF (in intact cells) or to guanosine 5'-(3-O-thio)triphosphate (in electrically permeabilized cells).

Manoalide, a natural sesterterpenoid that inhibits calcium channels.

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.

Loss of secretory response of rat basophilic leukemia (2H3) cells at 40 degrees C is associated with reversible suppression of inositol phospholipid breakdown and calcium signals.

Antigen-induced stimulatory signals as well as histamine secretion from the RBL-2H3 cells were found to be highly temperature dependent. There was no hydrolysis of inositol phospholipids, increase in cytosol calcium concentration (calcium signal), or secretion upon antigen stimulation at temperatures below 20 degrees C. At higher temperatures (i.e., 20 to 37 degrees C), all responses increased in extent with increase in temperature. Temperatures of 38 degrees C or higher, however, resulted in a marked decline in all responses, until no responses were observed at 40 to 42 degrees C. As indicated by the decay in calcium signal, the duration of response was also temperature dependent. The response was of long duration at 30 to 32 degrees C, but it became progressively more transient as the temperature was increased from 32 to 40 degrees C. The effects of low or high temperature were fully reversible. For example, in the presence of antigen, stimulatory signals immediately appeared once the temperature was decreased from 40 to 37 degrees C. Although the diminished responses could be explained, in part, by a reduction in rates of IgE receptor aggregation and phospholipase C activity, the reductions were insufficient to account for complete loss of activity at 40 degrees C. We conclude that generation of intracellular signals in 2H3 cells is blocked by quite small elevations in temperature above 37 degrees C, possibly as consequence of changes in membrane fluidity.