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The nomenclature used in the literature for the heterocyclic and exocyclic PAC is often diversified and confusing. Different preferences for naming generic classes and individual heterofunctional PAC are common. An increase in the number of papers presented in the PAH symposium proceedings on N-, O- and S-PAC has prompted a brief compilation of the key rules of nomenclature for these classes of compounds. Hence, a synopsis of the current rules for nomenclature applied to monofunctional N-, O-, and S-PAC as published by the International Union of Pure and Applied Chemistry (IUPAC) is presented here in an effort to clarify and unify nomenclature used in the proceedings of these symposia. The nomenclature methods that are abridged and presented in this paper were taken mainly from Sections A, B, and C of the IUPAC rules on nomenclature of organic chemicals. In this paper, the nomenclature for the nonfunctional PAH is first briefly reviewed, followed by three sections devoted to outlining the key aspects of naming the N-, O-, and S-PAC.

Receptor mediated calcium entry (RMCE) is a now recognised mechanism by which the influx of Ca 2+ into cells can occur. Agents which antagonise or block this mechanism are expected to possess anti-platelet aggregation and anti-inflammatory activities. The first series of RMCE blockers based upon the lead compound SC 38249, has been prepared. Their synthesis, activity against ADP evoked Ca 2+ signals in human platelets and their anti-platelet aggregation activity is described. A trend between the c log P of these compounds and their activity as inhibitors of Ca 2+ influx into human platelets stimulated by ADP was observed. This SAR study led to a 40-fold increase in activity over the initial lead compound.

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN′N′-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

Collagen (10-90 micrograms/ml) and ionomycin (1 microM; a calcium ionophore) each evoked rises in intracellular free calcium, protein kinase C activity and arachidonic acid release in human platelets, and as previously demonstrated for collagen, ionomycin (1 microM) stimulated protein tyrosine phosphorylation. However, at lower concentrations (60 and 250 nM) ionomycin selectively mobilised calcium. Ro31-8220 (a selective inhibitor of protein kinase C) inhibited (by 50%) ionomycin-stimulated arachidonic acid release. Genistein (an inhibitor of protein tyrosine kinases) also reduced by 50% ionomycin-stimulated arachidonic acid release. In combination, genistein and Ro31-8220 abolished ionomycin-stimulated arachidonic acid release. These findings show 1) that a rise in calcium is not sufficient, and 2) the activation of both protein kinase C and protein tyrosine phosphorylation is necessary, for full ionomycin-stimulated arachidonic acid release in human platelets.

Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins Iron previously identified blooms of the blue-green alga Microcystis aeruginosa.

Lipoxin A4 (LXA4), a lipoxygenase-derived metabolite of arachidonic acid, stimulated a dose-dependent elevation in cytosolic free Ca2+ concentration, [Ca2+]i, in fura-2-loaded human neutrophils, with an EC50 of 0.4-0.5 microM. The time for [Ca2+]i to peak was also dose-dependent. In the presence of extracellular Ca2+ (CaDT-PA added), the rise in [Ca2+]i was due to a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. In the absence of extracellular Ca2+, the rise in [Ca2+]i was due to release from internal stores, which then became depleted. No response to LXA4 was seen in the absence of divalent cation chelators (EGTA or DTPA); this is presumably because LXA4 forms an inactive complex with heavy metal cations. In the presence of extracellular Ca2+, LXA4 had no effect on the subsequent response of neutrophils to the chemotactic peptide fmetleu-phe (fmlp). In the absence of extracellular Ca2+, LXA4 dose-dependently reduced the subsequent response of neutrophils to fmlp; this is presumably because LXA4 discharges the store, and so reduces the amount of Ca2+ available for subsequent release by fmlp.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [(Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.

Measurement of cytoplasmic free calcium, [Ca2+]i, in single human endothelial cells has shown that low doses of the inflammatory mediator histamine evoke asynchronous repetitive spikes in [Ca2+]i whereas high doses cause a maintained elevated [Ca2+]i. We discuss possible regulatory mechanisms, and the potential physiological and pathological implications of such a frequency-modulated [Ca2+]i signalling system.

Both substance P and carbachol produced increases in inositol tris- and tetrakisphosphate and increased cytosolic free [Ca2+] in dispersed parotid acinar cells loaded with fura-2. The increase in [Ca2+]i in response to each agonist was due to a combination of mobilization of internal Ca2+ and entry of extracellular Ca2+. Kinetic studies of the initial response to substance P, and measurement of peak [Ca2+]i, demonstrated that the initial rapid rise in [Ca2+]i was due to both internal release and entry of Ca2+. Substance P could evoke a greater initial increase in [Ca2+]i and inositol trisphosphate than could carbachol. However, after 1 min in the presence of external Ca2+, the maintained [Ca2+]i level in response to substance P was considerably smaller than that seen with carbachol, an effect apparently due to homologous desensitization of the substance P receptor. The two agonists each produced a similar 4-5-fold increase in inositol tetrakisphosphate levels within 30 s; this level was maintained in the presence of carbachol, but decreased with substance P. Similarly, the level of inositol (1,4,5)-trisphosphate decreased after prolonged incubation with substance P. Thus, the maintained level of [Ca2+]i, and by deduction Ca2+ entry, correlated with the levels of inositol (1,4,5)-trisphosphate and inositol tetrakisphosphate; a result consistent with a possible role for these inositol phosphates in the control of receptor-mediated Ca2+ channels.

Stimulation of platelets with thrombin or parotid acinar cells with carbachol results in an increase in [Ca2+]i which is due to both release from internal stores and influx across the plasma membrane. In platelets, thrombin also stimulates Mn2+ entry into the cytosol; this is seen as a stimulated quench of fura-2 fluorescence. In the parotid, however, carbachol does not stimulate Mn2+ entry. This result suggests different mechanisms of stimulated divalent cation entry in the two cell types and could have important implications in the study of receptor-mediated Ca2+ entry mechanisms.

Both protein kinase C and Ca2+ may act in concert to bring about activation of secretion. This study examined the actions on pancreatic acini of ionomycin and phorbol dibutyrate, which selectively stimulate one or the other of these pathways; their stimulatory effects were compared with those of receptor agonists, such as carbachol and caerulein, which activate phospholipase C. The Ca2+ ionophore ionomycin produced a dose-dependent increase in amylase secretion and intracellular free Ca2+ (as measured by quin-2). The increase in amylase secretion elicited by carbachol or caerulein was accompanied by a small sustained increase in intracellular free Ca2+, following an initial peak. However, the elevation in intracellular free Ca2+ produced by these receptor agonists for a given level of amylase secretion was less than that observed with ionomycin. Phorbol dibutyrate stimulated amylase secretion by a mechanism that was independent of extracellular Ca2+, and no change in intracellular free Ca2+ was observed. Synergistic stimulatory effects of phorbol dibutyrate and ionomycin were observed, whether the phorbol ester was present before, or in combination with, ionomycin. Diacylglycerols containing unsaturated fatty acids (1,2-dioleoylglycerol and 1,3-dioleoylglycerol) also stimulated amylase secretion and exhibited synergistic effects on secretion with ionomycin. These findings suggest that complete activation of amylase secretion from the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-activated pathways.

Electrically permeabilized cells of rat parotid gland, prelabelled with [ 3 H]-inositol, synthesized [ 3 H]-inositol phosphates (IP 3 and IP 2 ) when stimulated with α 1 -adrenergic, muscarinic-cholinergic, and substance P receptoragonists. Non-hydrolyzable analogues of GTP (GTPγS and GppNHp) also stimulated [ 3 H]-IP 3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [ 3 H]-IP 3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP 3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca 2+ -mobilizing receptors to phospholipase C in parotid gland.

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.

The initial response of many cells to ‘Ca2+-mobilizing’ agonists is phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate to inositol trisphosphate (IP3) and diacylglycerol. It has been suggested, by analogy with receptor regulation of adenylate cyclase, that ‘Ca2+-mobilizing’ receptors may interact with a guanine nucleotide-binding protein (G protein) to regulate phospholipase C activity. Here we report increased accumulation of IP3 in response to caerulein or carbachol in electrically permeabilized rat pancreatic acinar cells. The stable analogues of GTP (guanosine 5′-[gamma–thio]trisphosphate and guanosine 5′-[beta, gamma-imido]triphosphate) stimulate IP3 accumulation and potentiate the effects of caerulein and carbachol. This synergism demonstrates an interaction between receptors, a G protein and phospholipase C. These responses are unaffected by pretreatment of the cells with pertussis or cholera toxins under conditions that produce substantial covalent modification of Gi and Gs, the proteins that couple receptors to adenylate cyclase. We therefore conclude that the G protein that couples receptors to phospholipase C in exocrine pancreas is probably neither Gi nor Gs; instead, we propose that a different G protein mediates this effect.

Human umbilical-vein endothelial cells stimulated with thrombin or histamine show an increase in [Ca2+]i (cytoplasmic free calcium concn.) that is maintained well above the basal pre-stimulated value as long as agonist and a source of extracellular Ca2+ are present. These results provide circumstantial evidence that agonists stimulate influx of Ca2+ across the plasma membrane and into the cytoplasm. Here, we have used Mn2+ as the extracellular bivalent cation which can bind to the fluorescent Ca2+ indicator fura-2 to quench its fluorescence completely. Human umbilical-vein endothelial cells were loaded with fura-2 and, in the presence of extracellular Mn2+, thrombin and histamine were shown to cause quenching of the intracellular dye. This result demonstrates conclusively that agonists can stimulate the influx of bivalent cations. Stimulated discharge of Ca2+ from intracellular stores and influx of Mn2+ were temporally resolved in the same cells to show that release of Ca2+ from intracellular stores clearly precedes influx. Influx of Mn2+ was also demonstrated when extracellular Mn2+ was added after agonist at a time when [Ca2+]i had fallen back to the basal value, showing that influx is not dependent on elevated [Ca2+]i.

Calcium-mobilizing receptors stimulate polyphosphoinositide hydrolysis, catalysed by phospholipase C, with the formation of intracellular messengers that then control the cytosolic Ca 2+ concentration and the activity of protein kinase C. Colin Taylor and Janet Merritt describe recent studies which suggest that a guanine nucleotide-dependent regulatory protein (G protein) couples these receptors to phospholipase C, a finding that presents a striking parallel with receptor regulation of adenylate cyclase.

Stimulation of human umbilical-vein endothelial cells by agonists such as histamine or thrombin promotes an influx of Ca2+, causing an increase in cytoplasmic free Ca2+ ([Ca2+]i) that is dependent on the continued presence of both agonist and extracellular Ca2+. This influx can also be clearly detected by using Mn2+ as a marker for Ca2+ entry, since Mn2+ quenches fura-2 fluorescence. The internal stores can be discharged in nominally Ca2+-free solution by stimulation for a brief period by 100 microM-histamine, with the stimulation being terminated by addition of 20 microM of the H1 antagonist mepyramine. After this (i.e. in the continuous presence of antagonist) a stimulated bivalent-cation influx can still be detected, as evidenced by the following observations: (a) addition of Mn2+ produces a stimulated quench, (b) addition of Ca2+ produces a transient rise in [Ca2+]i, (c) addition of 1 unit of thrombin/ml produces a much attenuated response unless the cells are exposed for a short period to 1 mM extracellular Ca2+. These results imply that stimulated bivalent-cation influx may be a direct consequence of the discharge of the internal Ca2+ stores rather than a direct consequence of the presence of agonist.

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.

Both substance P and carbachol produced increases in inositol tris- and tetrakisphosphate and increased cytosolic free [Ca2+] in dispersed parotid acinar cells loaded with fura-2. The increase in [Ca2+]i in response to each agonist was due to a combination of mobilization of internal Ca2+ and entry of extracellular Ca2+. Kinetic studies of the initial response to substance P, and measurement of peak [Ca2+]i, demonstrated that the initial rapid rise in [Ca2+]i was due to both internal release and entry of Ca2+. Substance P could evoke a greater initial increase in [Ca2+]i and inositol trisphosphate than could carbachol. However, after 1 min in the presence of external Ca2+, the maintained [Ca2+]i level in response to substance P was considerably smaller than that seen with carbachol, an effect apparently due to homologous desensitization of the substance P receptor. The two agonists each produced a similar 4-5-fold increase in inositol tetrakisphosphate levels within 30 s; this level was maintained in the presence of carbachol, but decreased with substance P. Similarly, the level of inositol (1,4,5)-trisphosphate decreased after prolonged incubation with substance P. Thus, the maintained level of [Ca2+]i, and by deduction Ca2+ entry, correlated with the levels of inositol (1,4,5)-trisphosphate and inositol tetrakisphosphate; a result consistent with a possible role for these inositol phosphates in the control of receptor-mediated Ca2+ channels.

Many ligands which bind to specific cell-surface receptors exert their intracellular effects via common signal-transduction pathways. One such pathway, used by a wide variety of hormones and neurotransmitters, involves perturbation of the metabolism of intracellular, inositol-containing compounds. The precise relationship between changes in inositol metabolism and other cellular responses to receptor activation are not yet fully understood. It is clear however, that two important consequences of receptor activation are an increase in the intracellular, steady-state levels of DG and Insl45P3, which are known to activate protein kinase C and discharge an intracellular Ca2+-pool respectively [1], [2], [3]. The activation of protein kinase C and an increase in the concentration of cytosolic free Ca2+ ([Ca2+]i) are, in turn, responsible for mediating a number of responses to external stimulation e.g. smooth muscle contraction or secretion of the contents of intracellular granules.

Carbachol-evoked rises in [Ca2+]i were measured in fura-2-loaded, rat parotid acinar cells. In suspensions of dissociated cells examined by dual wavelength excitation fluorimetry, a maximally effective concentration of carbachol produced a measured peak [Ca2+]i of 780 +/- 60 nM followed by a maintained elevation in the presence of 1 mM external Ca2+, and a peak of 630 +/- 95 nM followed by a return to resting values in the absence of external Ca2+. Stopped-flow, single wavelength fluorimetry was used to resolve the rising phase of the response. There was a dose-dependent lag of 70-220 ms before [Ca2+]i started to increase, and [Ca2+]i was maximal by 800-900 ms. These times were similar in the presence or absence of external Ca2+, although the initial rate of rise was faster in the presence of external Ca2+. These kinetics are consistent with a biochemical event, possibly phosphatidylinositol bisphosphate hydrolysis, mediating both internal release and Ca2+ entry, with a component of the initial rise being due to Ca2+ entry.

A fiberglass/epoxy composite material and the vacuum bag molding process were used to replace an existing steel battery box for the Marine Corps MK-48 ground vehicle. This item has been plagued with corrosion problems that directly impacts operational readiness, reliability, and life cycle costs. Quality control, chemical resistance, and mechanical tests were performed after selection of an appropriate material and process. Design and fabrication of several battery boxes has shown a weight savings of 13.2 kg (29.1 lbs). Six composite battery boxes were delivered to the Marine Corps (Quantico, VA) for field testing.

The possible role of calcium as a primary mediator in the control of prolactin secretion from normal pituitary cells was examined. Basal prolactin secretion, and secretion stimulated by thyrotrophin releasing hormone (TRH), raised K + or the calcium ionophore, A23187, were all dependent on the presence of extracellular Ca2+. The calcium channel antagonists, methoxyverapamil, cobalt and manganese, inhibited basal, TRH- and K+-stimulated prolactin secretion. In addition, prolactin secretion stimulated by a phosphodiesterase inhibitor, isobutylmethylxanthine, which increases cellular cyclic AMP, was inhibited by these Ca2+ antagonists. These observations indicate that Ca2+ may be the primary intracellular mediator in the control of prolactin secretion, with cyclic AMP having a secondary modulatory role on Ca2+ influx, probably on voltage-dependent Ca2+ channels. J. Endocr. (1984) 101, 319–325

Stimulation of platelets with thrombin or parotid acinar cells with carbachol results in an increase in [Ca2+]i which is due to both release from internal stores and influx across the plasma membrane. In platelets, thrombin also stimulates Mn2+ entry into the cytosol; this is seen as a stimulated quench of fura-2 fluorescence. In the parotid, however, carbachol does not stimulate Mn2+ entry. This result suggests different mechanisms of stimulated divalent cation entry in the two cell types and could have important implications in the study of receptor-mediated Ca2+ entry mechanisms.

A considerable amount of evidence exists to suggest that a rise in cytoplasmic free calcium concentration, [Ca2+]i, evokes morphological, metabolic and secretory responses in endothelial cells in response to stimulation by many inflammatory mediators. One of the most potent agents for stimulating the release of the vasodilators endothelial-derived relaxing factor (EDRF) and prostacyclin (PGI2) is the calcium ionophore A23187. However, the hypothesis that elevated [Ca2+]i is the physiological messenger is held with some caution since A23187-evoked rises in [Ca2+]i sufficient to evoke these responses can conceivably be far larger than those physiologically attained on stimulation with inflammatory mediators. With the development of the fluorescent [Ca2+] indicator dyes quin2, fura-2 and indo-1, increases in [Ca2+]. have been observed in endothelial cells from bovine or porcine aortae or from human umbilical vein in response to histamine, bradykinin, thrombin, adenine nucleotides and PAF. Furthermore, recent work has shown that these mediator-evoked rises in [Ca2+]i are both sufficient and necessary for the production and release of PGI2 (Hallam et al., 1988c). Our attention has now focussed on the mechanisms of Ca2+ movement and the sources of trigger Ca2+.

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.

A wide variety of receptors appear to be coupled to a phospholipase C (EC 3.1.4.3) that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5-IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase.

Previous studies of the early kinetics of rises in cytosolic free [Ca2+] in fura-2-loaded human platelets suggested that: (1) Ca2+ entry slightly preceded internal discharge with thrombin and other agonists known to promote inositol lipid hydrolysis; (2) with ADP, Ca2+ entry occurred without measurable delay and clearly preceded internal Ca2+ discharge. In the present work, Mn2+ added to the external medium was used as a marker for Ca2+ entry. By using an excitation wavelength of 360 nm, a quench of fura-2 can be followed to report Mn2+ entry without ‘contamination’ of the signal by changes in [Ca2+], because at this isosbestic wavelength Ca2+ does not alter fura-2 fluorescence. The present results show that, with thrombin stimulation, readily discernible Mn2+ entry starts after discharge of internal Ca2+ and is maintained for many minutes. With ADP, Mn2+ entry starts without measurable delay (less than 20 ms) and clearly precedes internal Ca2+ discharge. However, the enhanced Mn2+ permeability is only short-lived. These results, considered alongside previous data, point to the possible presence of at least three different receptor-mediated Ca2+-entry mechanisms in human platelets, one of which may include regulation by the ‘state of filling’ of this dischargeable Ca2+ store.

maximal concentrations of GTPyS, or the synergistic interactions between agonists and GTPyS are unaffected. These results suggest that, in exocrine pancreas, the G protein that couples receptors to phospholipase C is distinct from those involved in regulation of adenylate cyclase. We conclude that a parallel exists between receptorregulation of phospholipase C and adenylate cyclase; in each a G protein couples receptors to the effector enzymes. In exocrine pancreas, at least, distinct G proteins regulate each of the etfector enzymes, thus allowing for independent control of each signalling pathway. Supported by NIH grants DE 05764 and AM 28029.