Differential activation of human skin cells by platelet activating factor: stimulation of phosphoinositide turnover and arachidonic acid mobilization in keratinocytes but not in fibroblasts

Treatment of cultured adult human keratinocytes with platelet activating factor (PAF) resulted in a rapid, dose dependent accumulation of inositol phosphates. Inositol trisphosphate (IP3), inositol bisphosphate (IP2) and inositol phosphate (IP) were elevated within 15 seconds of exposure to PAF (1 microM). Lyso-PAF, phosphatidylcholine (PC) and lyso-PC had no effect on levels of inositol phosphates, indicating that the effect of PAF was specific. PAF also raised cellular 1,2-diacylglycerol content (2-fold) within two minutes of addition and stimulated mobilization of arachidonic acid (AA) and release of prostaglandin E2. In contrast, PAF did not stimulate phosphoinositide turnover or AA release in cultured dermal fibroblasts. These results suggest that the inflammatory effects of PAF in human skin result, at least in part, from its ability to directly activate keratinocytes and stimulate release of pro-inflammatory eicosanoids.     

Luteinizing hormone-releasing hormone enhances polyphosphoinositide breakdown in rat granulosa cells

A 2-min addition of LHRH to [3H]inositol-prelabeled rat granulosa cells in primary culture evoked significant increases in the accumulation of [3H]inositol phosphates, i.e. radiolabeled inositol monophosphate (IP), inositol diphosphate (IP2), and inositol triphosphate (IP3) levels increased to 210, 590 and 520%, respectively, when compared to control cultures. By contrast, addition of FSH failed to elicit such a response. The effect of LHRH was completely blocked by the concomitant presence of a specific LHRH antagonist. LHRH evoked increase in [3H]IP3 and [3H]IP2 accumulation as early as 30 sec, while the increase in [3H]IP became significant at 2 min. These data support the hypothesis that polyphosphoinositide breakdown may be an early step in the intracellular signal mechanism which mediates the action of LHRH.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.     

Proopiomelanocortin-derived peptides, phosphoinositides, cAMP, and aldosterone secretion

Since the intracellular messengers of various proopiomelanocortin-derived peptides remain ambiguous at best, we have investigated the possible involvement of phosphoinositide metabolism in aldosterone secretion evoked by alpha-MSH, beta-LPH, as well as ACTH in rat and calf adrenal glomerulosa cells. We have also examined the cAMP responses in the adrenal glomerulosa cells to alpha-MSH comparing it with those of ACTH. Our results showed that neither alpha-MSH, beta-LPH, nor ACTH increased inositol triphosphate (IP3) or other inositol phosphates in adrenal glomerulosa cells while increasing aldosterone secretion from the same cells. Angiotensin II, known to cause hydrolysis of the phosphoinositides, increased IP3 in these adrenal cells in a dose-dependent manner. Both ACTH and alpha-MSH raised the cAMP levels in the calf adrenal glomerulosa cells, although the magnitude of the increase of cAMP in response to ACTH was greater. These findings suggest that IP3 as a mediator of alpha-MSH- and beta-LPH-induced aldosterone secretion is not likely and other mediator(s) may be involved.     

Arachidonic acid stimulates phosphoinositide metabolism and catecholamine release from bovine adrenal chromaffin cells

Arachidonic acid (AA) evoked a dose-dependent increase in the accumulation of inositol phosphates in cultured bovine adrenal chromaffin cells, and this effect was specific for AA. AA also induced a rise in [Ca2+]i, but this rise was markedly reduced by removal of extracellular Ca2+. AA-induced accumulation of inositol phosphates was absolutely dependent on extracellular Ca2+, and nicardipine and nifedine partially reduced it but verapamil had no effect. Moreover, AA dose-dependently stimulated catecholamine release from chromaffin cells in the presence of ouabain, and this effect was specific for AA. AA-induced catecholamine release in the presence of ouabain was also inhibited by nicardipine and nifedipine but not by verapamil. Furthermore, the phospholipase C inhibitor neomycin inhibited the release. These results taken together suggest that AA stimulates catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca2(+)-dependent manner.     

ADH-dependent phosphoinositide signalling system and prostaglandin E production in the frog urinary bladder.

Antidiuretic hormone (ADH) stimulated formation of inositol 1,4,5-trisphosphate (IP3), 1,2-diaclyglycerol (DAG) and an increase of phosphatidylinositol 4,5-biphosphate (PIP2) breakdown in the frog urinary bladder 20 s after addition. ADH also increased the prostaglandin E (PGE) secretion into serosal medium 3.5-fold and the release of arachidonic acid (AA) from 1,2-DAG, which was intensified in the presence of DAG kinase inhibitor R59022. Neomycin sulphate (10(-5) M) from the serosal side blocked ADH-stimulated PIP2 hydrolysis, IP3 production and increased the hydro-osmotic response to ADH. It also inhibited the ADH-stimulated PGE production (55%) and release of AA from 1,2-DAG. This data suggest that PIP2 breakdown is involved in the mechanism of feedback regulation of ADH action and is associated with PGE production via (i) the increase of AA release from PIP2-generated 1,2-DAG and (ii) possible activation of phospholipase A2 by IP3-induced elevation of cytosol Ca2+.     

Size of the inositol 1,4,5-trisphosphate-sensitive calcium pool in guinea-pig hepatocytes.

Permeabilized hepatocytes accumulated 45Ca2+ into a non-mitochondrial pool when provided with ATP. 45Ca2+ efflux from this pool was revealed by removal of ATP with glucose and hexokinase or by inhibiting uptake with NaVO3. The effect of inositol 1,4,5-trisphosphate (IP3) on 45Ca2+ efflux from the pool was investigated. IP3 (5 microM) evoked a rapid increase in the rate of 45Ca2+ efflux. Kinetic analysis of the effect of IP3 indicated the existence of two distinct Ca2+ fractions within the pool; only one, accounting for about one-third of the ATP-dependent Ca2+ content of the pool, was responsive to IP3. The effect of IP3 on 45Ca2+ efflux from the non-mitochondrial pool does not require ATP, a finding that is inconsistent with a previous suggestion that this effect may be mediated by protein phosphorylation.     

Bradykinin inhibits potassium (M) currents in N1E-115 neuroblastoma cells. Responses resemble those in NG108-15 neuroblastoma x glioma hybrid cells

Application of bradykinin to voltage-clamped N1E-115 mouse neuroblastoma cells evoked sequential outward and inward membrane currents, accompanied by an increase and decrease of membrane conductance, respectively. Methacholine produced an inward current with a decreased conductance. The outward current response to bradykinin was imitated by intracellular inositol 1,4,5-trisphosphate (IP3). Bath application of phorbol dibutyrate induced an inward current and potentiated the response to IP3. We conclude that the response of these cells to bradykinin is identical to that of NG108-15 hybrid cells, and therefore may be attributed to the dual effects of inositol trisphosphate and diacylglycerol formed by hydrolysis of phosphatidylinositide.     

Microinjection of inositol 1,2-(cyclic)-4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, and inositol 1,4,5-trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium

Inositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium-regulated chloride channels but do not have a calcium-induced calcium release system. Microinjection of inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,2-(cyclic)-4,5-trisphosphate (cIP3), inositol 1,4,5-trisphosphate (IP3), or inositol 4,5-bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1-phosphate (IP1), inositol 1,3,4,5,6-pentakisphosphate (IP5), inositol 1,4-bisphosphate, or inositol 3,4-bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half-maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono- or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long-term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 much greater than IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

Thrombin-induced inositol trisphosphate production by rabbit platelets is inhibited by ethanol.

Ethanol has an inhibitory effect on some platelet functions, but the mechanisms by which it exerts this effect are not known. Using suspensions of washed platelets, we observed that ethanol (1-9 mg/ml) did not affect the aggregation of rabbit platelets stimulated with ADP (0.5-10 microM). When platelets were prelabelled with 5-hydroxy[14C]tryptamine, aggregation and secretion of granule contents in response to thrombin (0.01-0.10 unit/ml) were not inhibited by ethanol, but these responses to thrombin at lower concentrations (less than 0.01 unit/ml) were inhibited by ethanol (2-4 mg/ml). Platelets were prelabelled with [3H]inositol so that increases in inositol phosphates upon stimulation could be assessed by measuring the amount of label in these compounds. ADP-induced increases in IP (inositol phosphate) and IP2 (inositol bisphosphate) were not affected by ethanol. IP3 (inositol trisphosphate) was not changed by ADP or ethanol. Although ethanol did not affect the increases in IP, IP2 and IP3 caused by stimulation of platelets with thrombin at concentrations greater than 0.01 unit/ml, ethanol did inhibit the increases observed at 2 and 3 min in these inositol phosphates caused by lower concentrations of thrombin (less than 0.01 unit/ml). Since ADP did not cause formation of IP3 in rabbit platelets, and since no thromboxane B2 was detected in platelets stimulated with the lower concentrations of thrombin, it is unlikely that the inhibitory effect of ethanol in IP3 formation was due to effects on further stimulation of platelets by released ADP or by thromboxane A2. Ethanol may inhibit platelet responses to thrombin by inhibiting the production of the second messenger, IP3.     

Norepinephrine stimulates the direct breakdown of phosphatidyl inositol in rat tail artery.

When segments of rat tail artery were labeled with [3H]inositol and then stimulated with norepinephrine (NE), the inositol phosphates produced were primarily IP and IP2, together with a small but significant amount of Ins(1,4,5)P3 and a very small amount of Ins(1,3,4,5)P4. It has been unclear in many studies whether or not the relatively large levels of IP and IP2 produced in [3H]inositol-labeled tissue represent indirect products of phosphatidyl inositol(4,5)bis phosphate breakdown (through Ins(1,4,5)P3) or direct products of phosphatidyl inositol 4 monophosphate and phosphatidyl inositol breakdown. In order to answer this question tail artery segments were prelabeled with [3H]inositol and then permeabilized with beta escin and stimulated with norepinephrine and GTP gamma S, so that increases in IP, IP2, and Ins(1,4,5)P3 were still observed. If these permeable segments were stimulated with agonist in the presence of compounds known to inhibit Ins(1,4,5)P3 5-phosphatase, such as glucose 6P, (2,3)diphosphoglycerate, or Ins(1,4,5)P3, the levels of labeled Ins(1,4,5)P3 and labeled IP2 were increased, while the level of stimulated labeled IP was unchanged. This indicated that some of the IP2 and IP formed in these cells was produced from PIP2 but that some of these compounds might be formed from PIP or PI. When the isomers of inositol monophosphate, Ins 1P and Ins 4P, were separated by HPLC, it was shown that after prelabeled tail artery was stimulated by norepinephrine for periods of 1-2 min, the predominant isomer formed was Ins 4P, indicating either PIP2 or PIP as the source. However, after 5-20 min stimulation, both Ins 1P and Ins 4P were formed in equal amounts, suggesting that during sustained stimulation of smooth muscle PI itself was broken down directly. Therefore it appears that within 1-2 min of norepinephrine addition to vascular smooth muscle the bulk of the IP and IP2 produced are derived from PIP2 via IP3, while after 20 min of norepinephrine treatment much of the IP comes directly from PI. This suggests that the regulation of PLC in this tissue is more complicated than has been previously believed.     

Preparation of quality inositol pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP(5;)) and inositol hexakisphosphate (phytic acid or IP(6;)). IP(5;) and IP(6;) are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds(1). Phosphorylation of IP(6;) generates diphoshoinositolpentakisphosphate (IP(7;) or PP-IP(5;)) and bisdiphoshoinositoltetrakisphosphate (IP(8;) or (PP)(2;)-IP(4;)). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution(2-4). The IP(6;) kinases (IP(6;)Ks) posses an enormous catalytic flexibility, converting IP(5;) and IP(6;) to PP-IP(4;) and IP(7;) respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules(5,6). Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP(1;) (also referred as PP-IP(5;)K), which is able to convert IP(6;) to IP(7;) and IP(8;)(7,8). Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion(9), telomere length(10,11), chemotaxis(12), vesicular trafficking(13), phosphate homeostasis(14) and HIV-1 gag release(15). Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT(16). Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins(17). The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus(18,19). These procedures use acidic conditions that might lead to inositol pyrophosphate degradation(20) and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories. In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP(6;)-kinase and PP-IP(5;)-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates(20). Following IP(6;)K1 and PP-IP(5;)K enzymatic reactions using IP(6;) as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.     

Rapid functional assays of recombinant IP3 receptors

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).     

Gated conductances in native and reconstituted membranes from frog olfactory cilia.

Although cAMP is well established as a second messenger for olfactory transduction in vertebrates, the role of inositol 1,4,5-trisphosphate (IP3) in this process remains controversial. We addressed this issue by comparing currents evoked by cAMP and IP3 in native and reconstituted membranes from olfactory cilia. We detected only a cyclic nucleotide-gated conductance in the native membrane but both cyclic nucleotide-gated and IP3-gated conductances in the reconstituted membrane. The magnitudes of the cyclic nucleotide- and IP3-gated conductances were not correlated with each other in reconstituted membranes, suggesting that cyclic nucleotide- and IP3-gated channels originate in different cellular compartments.     

The interconversion of inositol 1,3,4,5,6-pentakisphosphate and inositol tetrakisphosphates in AR4-2J cells.

Data from several cell types have indicated that activation of hormone receptors promotes the metabolism of inositol 1,3,4,5,6-pentakisphosphate (IP5) to inositol 3,4,5,6-tetrakisphosphate ((3,4,5,6)IP4). However, to date, metabolism of IP5 by cell-free preparations has resulted in the formation of only inositol 1,4,5,6-tetrakisphosphate ((1,4,5,6)IP4). Thus, the metabolic relationships of IP5 with various inositol tetrakisphosphate (IP4) isomers have been investigated in both intact cells and cell homogenates of the rat pancreatoma cell line, AR4-2J. The steady-state concentration of IP5 was estimated to be 65 microM, while the combined concentration of (3,4,5,6)IP4 and (1,4,5,6)IP4 was approximately 1.0 microM. AR4-2J cell homogenates converted (1,3,4,6)IP4, (3,4,5,6)IP4, and (1,4,5,6)IP4 to IP5. (1,4,5,6)IP4 previously has not been demonstrated to be a precursor of IP5. To alter steady-state levels of inositol phosphates that were maintained by phosphorylation-dephosphorylation cycles, intact cells were treated with 10 microM antimycin A which reduced ATP levels by > 90% within 10 min. Following 2 h of treatment with antimycin A, there was a 6-fold increase in both (3,4,5,6)IP4 and (1,4,5,6)IP4, presumably derived from IP5. Experiments with cell-free systems determined that IP5 was dephosphorylated to (1,4,5,6)IP4 by a predominantly particulate Mg(2+)-independent, Li(+)-insensitive IP5 3-phosphatase. However, in the presence of 5 mM MgATP, IP5 also was metabolized to (3,4,5,6)IP4. Therefore, our data demonstrate novel and complex relationships between IP5, (3,4,5,6)IP4, and (1,4,5,6)IP4.     

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

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

Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production.

Isolated rat hepatocytes in primary monolayer culture were maintained for 18-24 h in the presence of 10% (v/v) serum and [3H]inositol. Vasopressin (100 nM) stimulated the production of inositol mono-, bis- and tris-phosphates (IP1, IP2, and IP3). Prior exposure of hepatocytes to 8-bromo cyclic AMP (8Br-cAMP; 100 microM), but not 8-bromo cyclic GMP, enhanced the vasopressin-mediated stimulation of inositol phosphate accumulation, but had no significant effect on their formation in the absence of vasopressin. The effect of the cyclic AMP analogue was mimicked by glucagon (10 nM), and was seen whether cyclic AMP or glucagon was added 5 min or 12 h before the addition of vasopressin. An 8 h incubation with dexamethasone (100 nM) enhanced the accumulation of IP3, but not that of IP2 or IP1, in the presence of 8Br-cAMP and vasopressin. Cycloheximide or actinomycin D had little effect on the vasopressin stimulation of inositol phosphate accumulation, after an 8 h incubation in the presence or absence of 8Br-cAMP.     

Internal perfusion of guinea-pig hepatocytes with buffered Ca2+ or inositol 1,4,5-trisphosphate mimics noradrenaline activation of K+ and Cl- conductances

External application of noradrenaline to voltage-clamped guinea-pig isolated hepatocytes evoked membrane conductance increases to K+ and Cl-. This effect was reproduced by internal perfusion of the cells with 2 microM buffered Ca2+ and with 20 microM inositol 1,4,5-trisphosphate (IP3). The kinetic properties of the K+ conductance and its selective block by the toxin apamin were the same in each case. Cyclical fluctuations of conductance observed with noradrenaline were reproduced by internal IP3 but not by Ca2+ perfusion, indicating that oscillations of intracellular free Ca2+ may arise from properties of the Ca2+ sequestration mechanism at constant IP3 concentration.     

Characteristics of Phorbol Ester- and Agonist-Induced Down-Regulation of Astrocyte Receptors Coupled to Inositol Phospholipid Metabolism

We have examined some of the characteristics of phorbol ester- and agonist-induced down-regulation of astrocyte receptors coupled to phosphoinositide metabolism. Our results show that preincubation of [3H]inositol-labelled astrocyte cultures with phorbol 12-myristate 13-acetate (PMA) resulted in a time- (t 1/2, 1-2 min) and concentration-dependent (IC50, 1 nM) decrease in the accumulation of [3H]inositol phosphates (IP) evoked by muscarinic receptor stimulation. Much longer (30-40 min) preincubation periods with higher concentrations (IC50, 600 microM) were required to elicit the same effect with the receptor agonist carbachol. Following preincubation, agonist-stimulated [3H]IP accumulation recovered with time; in both cases pretreatment levels of inositol lipid metabolism were attained within 2 days. Both phorbol ester and agonist pretreatments were also effective in reversing the carbachol-evoked mobilisation of 45Ca2+ in these cells. However, their effects on phosphoinositide metabolism were found not to be additive. Although neither pretreatment affected the incorporation of [3H]inositol into phosphoinositides, both resulted in a loss of membrane muscarinic receptors as assessed by [3H]N-methylscopolamine binding. In washed membranes prepared from [3H]inositol-labelled cultures, the guanine nucleotide analogue, guanosine 5'-O-thiotriphosphate (GTP-gamma-S), caused a dose-dependent increase in [3H]IP formation. This response was enhanced when carbachol was also included in the incubation medium, although the agonist alone was without effect. Pretreatment with either PMA or carbachol had no effect on GTP-gamma-S-stimulated [3H]IP accumulation but did reduce the ability of carbachol to augment this response. Similar findings were obtained when membranes were exposed directly to PMA.(ABSTRACT TRUNCATED AT 250 WORDS)