Basal Levels Of Inositol Phosphates Research Articles

Receptor Subtype-specific Docking of Asp6.59 with C-terminal Arginine Residues in Y Receptor Ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.

Contacts between Extracellular Loop Two and Transmembrane Helix Six Determine Basal Activity of the Thyroid-stimulating Hormone Receptor

A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.

The Detection of Micromolar Pericellular ATP Pool on Lymphocyte Surface by Using Lymphoid Ecto-Adenylate Kinase as Intrinsic ATP Sensor

Current models of extracellular ATP turnover include transient release of nanomolar ATP concentrations, triggering of signaling events, and subsequent ectoenzymatic inactivation. Given the high substrate specificity for adenylate kinase for reversible reaction (ATP + AMP <--> 2ADP), we exploited lymphoid ecto-adenylate kinase as an intrinsic probe for accurate sensing pericellular ATP. Incubation of leukemic T- and B-lymphocytes with [3H]AMP or [alpha-32P]AMP induces partial nucleotide conversion into high-energy phosphoryls. This "intrinsic" AMP phosphorylation occurs in time- and concentration-dependent fashions via nonlytic supply of endogenous gamma-phosphate-donating ATP, remains relatively resistant to bulk extracellular ATP scavenging by apyrase, and is diminished after lymphocyte pretreatment with membrane-modifying agents. This enzyme-coupled approach, together with confocal imaging of quinacrine-labeled ATP stores, suggests that, along with predominant ATP accumulation within cytoplasmic granules, micromolar ATP concentrations are constitutively retained on lymphoid surface without convection into bulk milieu. High basal levels of inositol phosphates in the cells transfected with ATP-selective human P2Y2-receptor further demonstrate that lymphocyte-surrounding ATP is sufficient for triggering purinergic responses both in autocrine and paracrine fashions. The ability of nonstimulated lymphocytes to maintain micromolar ATP halo might represent a novel route initiating signaling cascades within immunological synapses and facilitating leukocyte trafficking between the blood and tissues.

Inverse Agonist Activity of Sarpogrelate, a Selective 5-HT2A-Receptor Antagonist, at the Constitutively Active Human 5-HT2A Receptor

Mutations producing constitutively active G-protein coupled receptors have been found in the pathophysiology of several diseases, implying that inverse agonists at the constitutively active receptors may have preferred therapeutic applications. Because of the involvement of 5-HT2A receptors in mediating many cardiovascular diseases, constitutively active mutants of the 5-HT2A receptor may be responsible for the disease states. Thus, the purpose of the present study was to investigate the inverse agonist activity of sarpogrelate, a selective 5-HT2A-receptor antagonist, and its active metabolite, M-1; and we compared their activities with those of other 5-HT2A-receptor antagonists such as ritanserin, ketanserin, and cyproheptadine. Using a constitutively active mutant (C322K) of the human 5-HT2A receptor, we demonstrated that like other 5-HT2A-receptor antagonists, sarpogrelate acts as a potent inverse agonist by significantly reducing basal inositol phosphate levels. However, there were no significant differences between sarpogrelate and other 5-HT2A-receptor antagonists for their inverse agonist activity. Compared with the wild type receptor, mutant receptor displayed significantly higher affinity for 5-HT and lower affinity for sarpogrelate. These results indicate that stabilization of the inactive conformation of the 5-HT2A receptor may be a key component of the mechanism of action of sarpogrelate.

GPR80/99, proposed to be the P2Y15 receptor activated by adenosine and AMP, is not a P2Y receptor

The orphan receptor GPR80 (also called GPR99) was recently reported to be the P2Y15 receptor activated by AMP and adenosine and coupled to increases in cyclic AMP accumulation and intracellular Ca2+ mobilization (Inbe et al. J Biol Chem 2004; 279: 19790–9[12]). However, the cell line (HEK293) used to carry out those studies endogenously expresses A2A and A2B adenosine receptors as well as multiple P2Y receptors, which complicates the analysis of a potential P2Y receptor. To determine unambiguously whether GPR80 is a P2Y receptor subtype, HA-tagged GPR80 was either stably expressed in CHO cells or transiently expressed in COS-7 and HEK293 cells, and cell surface expression was verified by radioimmunoassay (RIA). COS-7 cells overexpressing GPR80 showed a consistent twofold increase in basal inositol phosphate accumulation. However, neither adenosine nor AMP was capable of promoting accumulation of either cyclic AMP or inositol phosphates in any of the three GPR80-expressing cells. A recent paper (He et al. Nature 2004; 429: 188–93 [15]) reported that GPR80 is a Gq-coupled receptor activated by the citric acid cycle intermediate, α-ketoglutarate. Consistent with this report, α-ketoglutarate promoted inositol phosphate accumulation in CHO and HEK293 cells expressing GPR80, and pretreatment of GPR80-expressing COS-7 cells with glutamate dehydrogenase, which converts α-ketoglutarate to glutamate, decreased basal levels of inositol phosphates. Taken together, these data demonstrate that GPR80 is not activated by adenosine, AMP or other nucleotides, but instead is activated by α-ketoglutarate. Therefore, GPR80 is not a new member of the P2Y receptor family.

Delta-opioid agonist-stimulated inositol phosphate formation in isolated, rabbit iris-ciliary bodies: role of G i/o proteins and Gβγ-subunits

Our laboratory has previously demonstrated the ability of kappa- and delta-opioid agonists to decrease aqueous flow rates and intraocular pressure of rabbits. The mechanisms by which these agents act in the ciliary body of the rabbit could involve inhibition of cAMP production, as well as increased generation of inositol phosphates (IPs). With regard to enhanced production of IPs, it has been suggested that their levels can be augmented by stimulation of phospholipase C via opioid receptors linked to either Gα-subunits derived from Gq proteins or Gβγ-subunits derived from G i/o-proteins. The aim of the current study is to investigate the role of pertussis toxin (PTX)-sensitive G-proteins and Gβγ-subunits in delta-opioid agonist-mediated changes in IP production in the isolated, rabbit iris-ciliary body (ICB). In one set of experiments, ICB segments were treated with the delta agonist, SNC80 (10 −12–10 −7 mol/l) alone. Other experiments were conducted utilizing SNC80 following pretreatment with either phosducin (Gβγ-subunit scavenger), PTX, or the delta antagonist, naltrindole. IP production was measured by ion exchange chromatography. Basal levels of IPs in the rabbit ICB were 58 287±2162, 15 218±969 and 2083±367 dpm/mg protein for IP 1, IP 2 and IP 3, respectively. The highly selective delta-opioid receptor agonist, SNC80, produced concentration-dependent increases in the levels of the IPs in the ICB, which were diminished in the presence of the delta antagonist, naltrindole, indicating the effect was mediated via activation of delta-opioid receptors. Pretreatment of tissues with PTX (75 ng/ml), completely abolished the concentration-dependent production of IPs generated by SNC80 (10 −11–10 −7 mol/l). In addition, pretreatment with phosducin (1 nmol/l) ablated the SNC80 (1 nmol/l)-induced increase in the formation of all three inositol phosphates. Results from this study indicate that the delta-opioid receptor-mediated increase in IP production is a PTX-sensitive G i/o response that involves participation of Gβγ-subunits. Thus, delta-opioid receptor activation by SNC80 in the ICB could be responsible, in part, for suppression of aqueous humor dynamics.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Constitutive activation of the neurotensin receptor 1 by mutation of Phe(358) in Helix seven.

1. The neurotensin receptor 1, NTS1, is a G protein-coupled receptor with seven transmembrane domains (TM) that mediates most of the known effects of the neuropeptide. Our previous studies have pointed to extracellular loop 3 and adjacent TM7 as being potentially involved in agonist-induced activation of the NTS1. 2. Here we investigated residues in these domains that might be involved in transconformational activation of the rat NTS1. Single amino acid mutated receptors were expressed in COS cells and inositol phosphate (IP) and cyclic AMP productions were studied. 3. The F358A mutation in TM7 resulted in a time- and receptor concentration-dependent increase in spontaneous IP production. At expression levels of 12 pmol mg(-1), agonist-independent IP production was increased 10 fold over basal for the F358A mutant receptor whereas the wild type NTS1 exhibited virtually no spontaneous activity at expression levels of 7.5 pmol mg(-1). 4. Neurotensin remained agonist on the F358A mutant receptor with a maximal effect that amounted to greater than twice basal IP levels. SR 48692 was inverse agonist at the mutant receptor, reversing IP production almost back to the levels measured in wild type NTS1-transfected cells. 5. Cyclic AMP production was not constitutively activated with the F358A mutant receptor but was stimulated by neurotensin with the same concentration dependence as that observed with the wild type NTS1. 6. This is the first report, to our knowledge, of a constitutively active mutant of the NTS1. The data are consistent with TM7 being involved in the transconformational changes that lead to agonist-induced coupling of the NTS1 to Gq.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Galpha16 mimics vasoconstrictor action to induce smooth muscle alpha-actin in vascular smooth muscle cells through a Jun-NH2-terminal kinase-dependent pathway.

Prolonged exposure of vascular smooth muscle cells (VSMC) to vasoconstrictors such as vasopressin or angiotensin II induces hypertrophy and increases expression of muscle-specific genes including smooth muscle alpha-actin (SM-alpha-actin). These vasoconstrictors signal through G-proteins, including members of the Gq family. To further investigate the role of Gq family members, VSMC were transfected with a constitutively active mutant of a Gq family member, Galpha16 (Galpha16Q212L). Stable expression of Galpha16Q212L persistently stimulated phospholipase C, resulting in increased basal levels of inositol phosphates. These cells were hypertrophied and expressed elevated levels of SM-alpha-actin compared with wild-type VSMC or cells transfected with a control plasmid (Neo). SM-alpha-actin promoter activity was markedly increased in cells stably or transiently expressing Galpha16Q212L. Basal c-Jun-NH2-terminal kinase (JNK) activity was increased 3-9-fold in cells stably expressing Galpha16Q212L, while basal activity of the p42/44 mitogen-activated protein kinases (ERKs) was unaffected. Transient expression of a kinase inactive JNK kinase partially inhibited induction of SM-alpha-actin promoter activity in response to vasoconstrictors or expression of Galpha16Q212L. These results indicate that expression of constitutively active Galpha16 in VSMC mimics the effects of vasoconstrictors on hypertrophy and muscle-specific gene expression, and activation of JNK may play a role in these responses.

TGF-β isoforms fail to modulate inositol phosphates and cAMP in normal and tumour-derived human oral keratinocytes

This study examined inositol phosphate and cAMP regulation by TGF-β1, -β2 and -β3 in normal and tumour-derived human oral keratinocytes. Previous findings indicated that the cell lines expressed TGF-β cell surface receptors and had a range of response to exogenous TGF-β1, -β2 and -β3 from being refractory to the ligand to marked inhibition. Basal levels of inositol phosphates broadly reflected the differentiation status of the cells as demonstrated by involucrin expression, but did not correlate with responsiveness to TGF-β1, as measured previously by thymidine incorporation. Treatment of cells with bradykinin or serum caused up-regulation of inositol phosphate levels; by contrast, TGF-β1, -β2 and -β3 failed to modulate inositol phosphates. In two tumour-derived cell lines, the TGF-β isoforms had no effect on cAMP levels, despite a significant increase in cAMP using a potent agonist of adenylate cyclase (forskolin). Furthermore, the cAMP analogue, dibutyryl cAMP, failed to mimic the inhibitory or refractory responses of TGF-β in these cell lines. The results demonstrate that in normal and tumour-derived human oral keratinocytes, TGF-β signal transduction is not mediated by inositol phosphates or cAMP.

Pharmacological selectivity of the cloned human P2U‐purinoceptor: potent activation by diadenosine tetraphosphate

1. The human P2U-purinoceptor was stably expressed in 1321N1 human astrocytoma cells and the pharmacological selectivity of the expressed receptor was studied by measurement of inositol lipid hydrolysis. 2. High basal levels of inositol phosphates occurred in P2U-purinoceptor-expressing cells. This phenomenon was shown to be due to release of large amounts of ATP from 1321N1 cells, and could be circumvented by adoption of an assay protocol that did not involve medium changes. 3. UTP, ATP and ATP gamma S were full and potent agonists for activation of phospholipase C with EC50 values of 140 nM, 230 nM, and 1.72 microM, respectively. 5BrUTP, 2C1ATP and 8BrATP were also full agonists although less potent than their natural congeners. Little or no effect was observed with the selective P2Y-, P2X-, and P2T-purinoceptor agonists, 2MeSATP, alpha,beta-MeATP, and 2MeSADP, respectively. 4. Diadenosine tetraphosphate, Ap4A, was a surprisingly potent agonist at the expressed P2U-purinoceptor with an EC50 (720 nM) in the range of the most potent P2U-purinoceptor agonists. Ap4A may be a physiologically important activator of P2U-purinoceptors.

Interaction of organophosphorus compounds with muscarinic receptors in SH‐SY5Y human neuroblastoma cells

Human neuroblastoma cells (line SH-SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH-SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phosphorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'-diisopropyl phosphorodiamide fluoridate) that ranged between 10(-9) M and 10(-3) M (10(-2) M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]-N-methylscopolamine (3H-NMS). Concentrations of paraoxon > 0.5 x 10(-3) M and PSP 1 x 10(-3) M significantly inhibited the binding of a saturating concentration of 3H-NMS. Concentrations of > 10(-5) M paraoxon or PSP could significantly inhibit the binding of a half-saturating concentration of 3H-NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX-116 for M2, and 4-DAMP for M3) indicated that SH-SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10(-8) M for 4-DAMP compared to 2.5 x 10(-6) M and 2.7 x 10(-5) M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phosphoinositides the capability of OPs to alter levels of inositol phosphates and agonist-stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo-inositol and then incubated for 15 min with the OPs before addition of 10(-5) M to 10(-3) M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration-related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of inositol phosphates that may be associated with another site of action in SH-SY5Y cells.

Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors.

The molecular mechanisms through which muscarinic receptors are activated upon binding of the neurotransmitter acetylcholine (ACh) are still poorly understood. Classical structure-function relationship studies have previously established that the ACh ester moiety plays a key role in muscarinic receptor recognition and activation. Consistent with this notion, all recently proposed three-dimensional muscarinic receptor models predict that an asparagine residue present in transmembrane domain VI of all muscarinic receptors is critically involved in the binding of the ACh ester moiety by means of hydrogen bonding. To test the correctness of this hypothesis, we created several mutant m3 muscarinic receptors in which this residue (Asn507) was replaced with alanine, serine, or aspartic acid. Radioligand binding studies with transfected COS-7 cells showed that, in contrast to the predictions made based on molecular modeling studies, all three mutant receptors were able to bind ACh and the structurally related muscarinic agonist, carbachol, with high affinities which differed from the corresponding wild type values by less than 5-fold. However, all three mutations led to dramatic reductions (235-28,300-fold) in binding affinities for certain subclasses of muscarinic antagonists including atropine-like agents and pirenzepine. The m3(Asn507-->Ala) and m3(Asn507-->Asp) mutant receptors were able to mediate carbachol-induced phosphatidylinositol hydrolysis in a fashion similar to that of the wild type receptor. Interestingly, the m3(Asn507-->Ser) mutant receptor displayed about 2-fold increased basal inositol phosphate levels, raising the possibility that it is constitutively active. In conclusion, our data suggest that the asparagine residue present in transmembrane domain VI of all muscarinic receptors is not critical for ACh binding and agonist-induced receptor activation, but plays a key role in the binding of certain subclasses of muscarinic antagonists.

Altered protein kinase C regulation of phosphoinositide-coupled receptors in deoxycorticosterone acetate-salt hypertensive rats.

This study examined the contribution of phosphatidylinositol metabolism and the efficacy of protein kinase C-mediated desensitization in the exaggerated alpha 1b-adrenergic receptor-mediated inositol phosphate response in the aorta of the deoxycorticosterone acetate (DOCA)-salt rat model of hypertension. The basal accumulation of inositol phosphates and the basal incorporation of [3H]myo-inositol in the phosphatidylinositol lipid pool were significantly higher in the aorta of these hypertensive rats. A positive correlation (r = .88, P < .01) was demonstrated between basal inositol phosphate levels and the [3H]myo-inositol-labeled phosphatidylinositol lipid pool. In hypertensive rats, alpha 1b-adrenergic receptor-mediated inositol phosphate production in response to phenylephrine was significantly higher compared with normotensive rats. Despite the normalization of phenylephrine-mediated inositol phosphate production to the [3H]myo-inositol-labeled phosphatidylinositol lipid pool, the alpha 1b-adrenergic response remained significantly higher in the hypertensive rats. Phorbol ester activation of protein kinase C attenuated to a lesser extent phenylephrine-mediated inositol phosphate production (40%) in the aorta of hypertensive rats compared with the 80% attenuation observed in the aorta of normotensive rats. This desensitization was inhibited in both groups by the protein kinase C inhibitor staurosporine. The blunted desensitization of the alpha 1b-adrenergic receptor by protein kinase C activation was not associated with a decrease in protein kinase C activity in the hypertensive rats, because aortic strips from these animals were more responsive to phorbol ester activation than aortic strips from normotensive animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogenesis of quisqualate-associated phosphoinositide metabolism in various regions of the rat nervous system

The effect of postnatal age on phosphoinositide metabolism per se and on quisqualate-stimulated phosphoinositide metabolism was characterized in synaptoneurosomes prepared from nine different regions of the rat nervous system, namely the brainstem, cerebellum, cerebral cortex, colliculi, hippocampus, hypothalamus, olfactory bulb, spinal cord and striatum. In the hippocampus, striatum, cerebellum, cerebral cortex, brainstem, colliculus and spinal cord, the basal levels of inositol phosphate (inositol-1-phosphate+inositol-4,5-bisphosphate) formation were maximal two days after birth and declined steeply to steady-state levels from the age of 10 postnatal days. Similarly, in the olfactory bulb, basal inositol phosphate synthesis did not significantly change when measured during the period from postnatal day 10 to 42. The extent of [3H]-inositol labelling of phosphoinositides as a function of age presented similar profiles when measured in hippocampal, striatal, cerebellar and cerebral cortical synaptoneurosomes, i.e. maximal at perinatal ages and minimal at adult ages. In the hypothalamus, [3H]-inositol labelling of phosphoinositides showed an increase from postnatal day 12 to higher levels from postnatal days 14 to 18 subsequently followed by a dramatic increase from postnatal day 21 to 42. A similar developmental trend was also obtained for basal inositol phosphate synthesis.On the whole, four types of developmental profiles for quisqualate-stimulated inositol phosphate formation (expressed as the percentage of the basal level and as the difference between stimulated and basal levels of radioactive inositol phosphates) were obtained depending on the nervous system region studied. In the early, prenatally developed nervous system regions, namely the brainstem and the spinal cord, no postnatal stimulation peaks of quisqualate-induced inositol phosphate formation were recorded. This was also the case for the colliculi when the stimulation of IP formation was expressed as the difference in basal and stimulated levels of inositol phosphates. Secondly, in the olfactory bulb a region known to possess a continuous capacity for developmental plasticity both structurally and functionally during the first three weeks of postnatal development, a simultaneous sustained high level of quisqualate stimulation of phosphoinositide metabolism (fluctuating around 200% of the basal level) during the early postnatal period was evident. Thirdly, in regions of the central nervous system like the cerebellum, cerebral cortex, hippocampus and the striatum known to undergo intense developmental activity during the first two postnatal weeks, peaks of quisqualate-stimulated phosphoinositide metabolism were initially detected around the first week after birth in each of these brain areas. Finally, in the hypothalamus where structurally unique postnatal developmental processes are known to take place, quisqualate-induced inositol phosphate formation progressively increases with age to reach maxima at postnatal day 18. The transient increases in quisqualate responses in the cerebellum, hippocampus and striatum are probably specific to quisqualate since carbachol-stimulated phosphoinositide metabolism yielded different age-associated response patterns. Similar increases of carbachol- and quisqualate-mediated phosphoinositide hydrolysis were on the other hand assayed in cerebral cortical and hypothalamic synaptoneurosomes. EC50, values for quisqualate (the quisqualate concentration required to produce 50% of the maximal effect) at postnatal days 6 and 10 were not significantly different in each of four types of synaptoneurosomes: cerebellar, cerebral cortical, hippocampal and striatal. On the basis of these latter results, it was deduced that the peak of quisqualate-stimulated phosphoinositide metabolism does not materialize on the basis of changes in quisqualate metabotropic receptor affinity. In conclusion, the measurement of inositol phosphate formation in synaptoneurosomes prepared from different regions of the postnatally developing nervous system indicate that there is a temporal correlation between the increased activity of quisqualate-stimulated phosphoinositide metabolism mediated by specific metabotropic glutamate receptors and region-specific developmental events. This could suggest a key role for certain metabotropic glutamate receptors in the developmental plasticity of the nervous system.

Epidermal growth factor potentiates the transmitter-induced stimulation of C-AMP and inositol phosphates in human pigment epithelial cells in culture

Salbutamol, isoproterenol and dopamine stimulate C-AMP production in human retinal pigment epithelium (RPE) cells by activation of β 2-type receptors. Epidermal growth factor (EGF) in contrast does not alter basal levels of C-AMP but elevates in an apparently dose-dependent manner the isoproterenol-induced stimulation of C-AMP. EGF also potentiates the forskolin-induced stimulation of C-AMP but has no effect on the elevation of C-AMP caused by NECA (5′-[ N-ethyl]-carboxamido adenosine), an adenosine A 2-receptor agonist. EGF, isoproterenol and NECA have no effect on basal levels of inositol phosphates (InsPs) in human RPE cells, but EGF specifically elevates the carbachol-induced stimulation of InsPs. The carbachol effect on InsPs is attenuated by the phorbol ester PMA (4β-phorbol 12 myrisate 13-acetate). PMA did not, however, affect the stimulation of C-AMP caused by isoproterenol. The interaction of EGF and C-AMP is further demonstrated in experiments where the incorporation of [ 3H]thymidine into RPE cells was studied, as an index for proliferation. EGF stimulates RPE cell proliferation while isoproterenol and dibutyryl C-AMP nullify the EGF effect. Dibutyryl C-AMP has a negative effect on RPE cell proliferation while isoproterenol is ineffective. The data presented here suggest that after stimulation of EGF receptors, tyrosine-kinase-activated products can influence secondary messenger products produced from activation of β 2-type (linked with C-AMP formation) and muscarinic (linked with InsPs production) receptors in RPE cells. We could find no evidence of an interaction between receptors associated with C-AMP and InsPs/diacylglycerol production.

Modulation of luteinizing hormone-stimulated inositol phosphate accumulation by phorbol esters in bovine luteal cells.

The present studies were conducted to evaluate the effects of protein kinase C activators on the inositol phospholipid-phospholipase C second messenger system in isolated bovine luteal cells. This report describes the effects of phorbol esters on inositol phosphate accumulation in LH- and prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea of early pregnancy were dispersed with collagenase and luteal cells were prelabelled for 3 h with [3H]inositol. Inositol phosphates produced in response to LH or PGF2 alpha were analyzed by ion exchange column chromatography. The tumor promoter and protein kinase C activator 12-O-tetradecanolyphorbol 13-acetate (TPA) had no effect on basal levels of inositol phosphates but inhibited LH-stimulated accumulation of inositol mono-, bis-, and trisphosphates by 72%, 68%, and 65%, respectively. TPA reduced the response to maximally effective concentrations of LH and tripled the concentrations of LH required to evoke half-maximal accumulation of inositol mono-, bis-, trisphosphates. The inhibitory effects of TPA were rapid (5 min) whether added before or after treatment with LH. Treatment with TPA also reduced (58%) the initial phase of intracellular calcium mobilization in LH-treated cells. The inhibitory effects of TPA were not associated with acute reductions in [3H]inositol incorporation, [3H]inositol phospholipid levels, cAMP levels, or progesterone accumulation in control or LH-stimulated luteal cells. The effects of phorbol esters were concentration dependent and specific for active tumor promoters with 10-50 nM TPA producing maximal inhibitory effects. A synthetic diacylglycerol, 1-oleyl-2-acetylglycerol, mimicked the inhibitory effects of TPA. In contrast, pretreatment with a physiological activator of protein kinase C, PGF2 alpha, had no effect on LH-stimulated inositol phosphate accumulation. The inhibitory effects of TPA could not be explained by a generalized inhibition of phospholipase C or G-proteins since the accumulation of inositol phosphates in PGF2 alpha- and NaF-treated cells was not inhibited by TPA. These results demonstrate that tumor promoting phorbol esters modulate the inositol phospholipid-phospholipase C transmembrane signaling system in LH-stimulated bovine luteal cells. The results suggest that phorbol esters may alter the coupling of the LH-receptor complex to phospholipase C. These findings implicate protein kinase C in the regulation of transmembrane signaling in the bovine corpus luteum.

Dopamine Inhibits Inositol Phosphate Production, Arachidonic Acid Formation, and Corticosteroid Release by Frog Adrenal Gland through a Pertussis Toxin-Sensitive G-Protein*

We have previously shown that dopamine-evoked inhibition of corticosteroid production from adrenocortical cells is mediated through a decrease in prostaglandin biosynthesis. Since the catecholamine did not alter the stimulatory effect of arachidonic acid, it was proposed that dopamine may inhibit the formation of arachidonate from glycerophospholipids. To test this hypothesis, the effect of dopamine on phosphoinositol lipid metabolism was investigated in frog interrenal (adrenal) tissue. In [3H]myo-inositol-prelabeled frog interrenal slices, a short pulse of dopamine (50 microM) induced a biphasic effect on inositol phosphate production: a transient (1-min) increase, followed by a sustained inhibition. Concurrently, dopamine induced a transient reduction followed by a sustained increase in polyphosphoinositides. A 10-min pulse of the D2 dopamine receptor agonist apomorphine (50 microM) elicited a significant inhibition of basal levels of inositol phosphates (tris-, bis-, and mono-), and an increase in plasma membrane phosphoinositol lipid contents. The inhibitory effect of dopamine on inositol phosphate formation and corticosteroid release was abolished by a 24-h incubation of interrenal slices with pertussis toxin. In [3H]arachidonic acid-prelabeled interrenal slices, dopamine also decreased diacylglycerol (DG) and arachidonic acid (AA) concentrations. A delay of 1 min was observed between inhibition of DG and arachidonate, suggesting that AA is probably generated from DG. We conclude that in the adrenal cortex, activation of dopamine D2 receptors is coupled to a phosphoinositide-specific phospholipase-C mediated via a pertussis toxin-sensitive G-protein. Taken together, our data indicate that inhibition of inositol phosphate and AA formation is one of the mechanisms by which dopamine controls corticosteroid production by adrenocortical cells.

P 2-purinoceptor-activated membrane currents and inositol tetrakisphosphate formation are blocked by suramin

The effect of suramin on the ATP-induced response in vas deferens DDT 1 MF-2 smooth muscle cells was studied. Stimulation of P 2-purinoceptors by ATP caused a change in membrane currents, measured by using the whole-cell patch-clamp configuration, and enhanced the formation of inositol phosphates, as analysed by high performance liquid chromatography. The ATP-induced membrane current consisted of a triphasis response, carried by a fast inward current, followed by a transient outward current and a sustained inward current. Inositol tetrakisphosphate (InsP 4) formation increased in the presence of ATP. The formation of the isomers Ins(1,3,4,5)P 4, Ins(1,3,4,6)P 4 and Ins(3,4,5,6)P 4 increased significantly after 5 min stimulation with ATP. Suramin inhibited the ATP-evoked membrane currents and the ATP-induced formation of inositol tetrakisphosphate isomers concentration dependently, but did not affect the basal inositol phosphate levels in the absence of ATP. These results indicate that suramin inhibits ATP-activated cellular processes in DDT 1 MF-2 vas deferens cells, most likely by acting on P 2-purinoceptors.