N-linked glycosylation is required for optimal AT1a angiotensin receptor expression in COS-7 cells.

The nature and role of glycosylation in AT1 angiotensin receptor (AT1-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT1a-Rs (HA-AT1a-Rs) in COS-7 cells. All three asparagine residues (Asn4, Asn176, Asn188) contained within consensus sites for N-linked glycosylation could be glycosylated in Cos-7 cells and appeared to be glycosylated on the endogenous AT1-R in bovine adrenal glomerulosa cells. Heterogeneity of glycosylation at each site accounted for the broad migration pattern of the AT1-R in SDS-PAGE. Mutation at each glycosylation site, either alone or in combination, had little effect on ligand binding parameters (although the N4K mutant had higher affinity) or signaling activity. However, an increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decreased surface expression of mutant HA-AT1a-Rs was correlated with decreased total cell receptor content as revealed by immunoblotting with an anti-HA antibody. These findings suggest that glycosylation enhances receptor stability, possibly by protecting nascent receptors from proteolytic degradation.

Raf-1 kinase activation by angiotensin II in adrenal glomerulosa cells: roles of Gi, phosphatidylinositol 3-kinase, and Ca2+ influx.

Little is known of the mechanisms leading to mitogen-activated protein kinase (MAPK) activation via Gq-coupled receptors. We therefore examined the pathways by which angiotensin II (Ang II) activates Raf-1 kinase, an upstream intermediate in the pathway to MAPK, via the Gq-coupled AT1 angiotensin receptor in bovine adrenal glomerulosa (BAG) cells. Ang II caused a rapid and transient activation of Raf-1 that reached a peak at 5-10 min. Ang II was a potent stimulus of Raf-1 activation with an ED50 of 10 pM and a maximal response at 1 nM, although higher Ang II concentrations elicited a submaximal response. Ang II-stimulated Raf-1 activity was unaffected by down-regulation of protein kinase C and intracellular Ca2+ chelation (using BAPTA) but was partially inhibited by pertussis toxin, and was abolished by manumycin A. Removal of extracellular Ca2+ (by EGTA) or blockade of L type Ca2+ channels (by nifedipine), as well as inhibition of MEK-1 kinase (by PD98059), enhanced Raf-1 activity, whereas wortmannin (100 nM) inhibited approximately one half of Ang II-stimulated Raf-1 activity. Hence, Raf-1 kinase activation by Ang II in BAG cells is dependent on Ras, is mediated in part via Gi and phosphatidylinositol 3-kinase, and is negatively regulated via Ca2+ influx and a downstream signaling element(s).

Agonist-induced phosphorylation of the endogenous AT1 angiotensin receptor in bovine adrenal glomerulosa cells.

A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.

Properties of AT1a and AT1b angiotensin receptors expressed in adrenocortical Y-1 cells.

Adrenocortical Y-1 cells were stably transfected with the AT1a and AT1b subtypes of the rat angiotensin (ANG)IIAT1 receptor cDNA to study the pharmacological and functional properties of the two receptors. Selected clones of transfected cells expressing the AT1a or AT1b receptor subtypes bound the native ligand ANG II and the peptide antagonist [Sar1,Ile8]ANG II with similar affinities, but they differed in their relative affinities for the nonpeptide antagonist losartan (half-maximal inhibitory concentration 9.7 and 4.7 nM), ANG III (126 and 33 nM), and the peptide antagonist [Sar1,Gly8]ANG II (6.2 and 1.2 nM). Photoaffinity labeling of the expressed receptors revealed a single component of 65 kDa for both receptor subtypes, suggesting that both receptors were glycosylated in a similar manner. The sensitivity of 125I-ANG II binding to AT1a and AT1b receptors to guanine nucleotides was unaffected by pertussis toxin treatment. ANG II stimulated the formation of inositol phosphates and increased the level of cytoplasmic Ca2+ in both At1a- and AT1b-transfected Y-1 cells. However, ANG II had little effect on forskolin-induced adenosine 3',5'-cyclic monophosphate accumulation, causing only minor inhibition in AT1a-transfected cells and slight enhancement in AT1b-transfected cells. These data indicate that AT1a and AT1b receptors show small but significant differences in their binding pharmacology and, upon activation, are coupled through Gq/G11 to the phosphoinositide-Ca2+ signaling pathway. However, neither AT1a nor AT1b receptors exhibit coupling to Gi and inhibition of adenylate cyclase when expressed in murine adrenal tumor cells.

A conserved NPLFY sequence contributes to agonist binding and signal transduction but is not an internalization signal for the type 1 angiotensin II receptor.

A conserved NPX2-3Y sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn298 and was reduced by replacement of Pro299 or Tyr302. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar1,Ile8]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe301, Tyr302, and Phe304, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe301 residue contributes significantly to agonist binding, and Asn298 is required for normal receptor activation and signal transduction.

Growth responses to angiotensin II in bovine adrenal glomerulosa cells.

The effects of angiotensin II (ANG II) on growth responses of primary cultures of bovine adrenal glomerulosa cells were studied to explore the mechanism(s) by which ANG II leads to hyperplasia and hypertrophy of the glomerulosa layer in sodium deficiency. ANG II did not increase [3H]thymidine incorporation during the first 5 days of culture, but mitogenic responses to ANG II became evident after longer periods of culture and were most prominent between 8 and 11 days after seeding. At this time, cell cycle analysis showed that ANG II increased the proportion of cells in the S phase and did not cause accumulation of cells in the G2 phase. Consistent with this finding, ANG II also stimulated proliferation of glomerulosa cells during treatment for 3 days in the presence of 1% serum. The mitogenic effect of ANG II was not inhibited by pretreatment with pertussis toxin and was mediated by AT1 receptors as indicated by its sensitivity to the subtype-selective antagonist DuP-753. Also, there was no emergence of AT2 receptors in glomerulosa cells during prolonged culture. These results indicate that intracellular mechanisms that mediate growth responses become more active during prolonged culture of glomerulosa cells. Thus, in addition to regulating the steroidogenic and secretory functions of the zona glomerulosa, ANG II exerts mitogenic actions that depend on the functional state of the glomerulosa cells.

Evidence for participation of calcineurin in potentiation of agonist-stimulated cyclic AMP formation by the calcium-mobilizing hormone, angiotensin II.

Angiotensin II (AII) receptors are known to interact with two distinct guanine nucleotide binding proteins, Gq/11 and Gi, in rat adrenal glomerulosa cells to activate phospholipase C and to inhibit adenylate cyclase, respectively. However, in cultured bovine glomerulosa cells AII potentiates rather than inhibits the stimulatory effect of adrenocorticotropin (ACTH) on cAMP levels. This effect of AII was partially mimicked by phorbol 12-myristate 13-acetate (PMA) and was partially inhibited by staurosporine or depletion of protein kinase C but was unaffected by pertussis toxin treatment. No potentiation was detectable in disrupted cells or in membrane preparations. In intact glomerulosa cells, treatment with cyclosporin A or FK506 completely inhibited AII- or PMA-induced potentiation of cAMP production without affecting the response to ACTH. In COS-7 cells transfected with the rat AT1 receptor, AII caused 2-3-fold enhancement of the ACTH-induced cAMP response, an effect that was partially reproduced by PMA. These potentiating actions of AII and PMA were prevented by preincubation with cyclosporin A or FK506, and the latter effect was abolished by rapamycin. These results implicate the Ca2+- and calmodulin-dependent protein phosphatase, calcineurin, in AII-induced enhancement of adenylate cyclase activity in both adrenal glomerulosa and transfected COS-7 cells. The finding that AII enhances ACTH-stimulated production of cAMP by a second messenger-mediated mechanism that involves the participation of calcineurin reveals an additional mode of cross-talk between pathways activated by Ca(2+)-mobilizing and cAMP-generating receptors.

Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction.

The relationship between angiotensin II-induced activation of G proteins and receptor internalization was analyzed by transiently expressing mutant and wild type cDNAs for the rat AT1a receptor in COS-7 cells. Pertussis toxin-sensitive G proteins did not appear to play a role in endocytosis since the receptor showed normal internalization kinetics in pertussis toxin-treated cells. Three deletion mutants of the third cytoplasmic loop revealed that the N-terminal part of this region is important for both receptor endocytosis and intracellular signaling. Three point mutations of Asp74, which has been implicated in signal transduction by the AT1a receptor, caused impaired G protein coupling and inositol phosphate responses. However, each of these mutants (D74N, D74H, and D74Y) showed markedly different internalization kinetics. The D74Y mutant showed the greatest impairment of internalization but retained the highest degree of inositol phosphate stimulation. In contrast, the D74N mutant, which showed the most impaired G protein coupling and inositol phosphate responses, had similar internalization kinetics to the wild type receptor. The combined mutant receptor containing the D74N substitution and deletion of residues 221-226 from the third cytoplasmic loop showed no G protein coupling or inositol phosphate response but was internalized about 60% as rapidly as the wild type receptor. These data demonstrate that endocytosis of the AT1 receptor is independent of agonist-activated signal transduction and indicate that receptor internalization and activation of phospholipase C have different structural requirements.

Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts.

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry.

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin II receptor subtypes and biological responses in the adrenal cortex and medulla.

Angiotensin II (AII) receptor subtypes and their potential coupling mechanisms were studied using recently developed peptide and nonpeptide antagonists in rat and bovine adrenal zona glomerulosa cells, as well as in membranes prepared from rat and bovine adrenal cortex and medulla. Comparison of the potencies of these novel antagonists to displace 125I-[Sar1,Ile8]AII from its binding sites revealed two distinct AII binding sites in membranes prepared from rat adrenal capsules (zona glomerulosa) and from rat adrenal inner zones containing the medulla. About 85% of the binding sites of the glomerulosa zone and 30% of those of the inner zones were of the AT1 subtype, with relative affinities for the nonpeptide antagonists Dup 753 and PD 123177 and the peptide antagonist CGP 42112A in the order of Dup 753 much greater than CGP 42112A greater than PD 123177. In contrast, the relative binding potencies for the other (AT2) population of binding sites were CGP 42112A greater than PD 123177 much greater than Dup 753. Neither AII nor its peptide antagonist [Sar1,Ile8]AII could distinguish between the two sets of binding sites. The effects of the new antagonists on functional responses of rat adrenal glomerulosa cells demonstrated that both AII-stimulated aldosterone production and the AII-induced inhibition of adrenocorticotropic hormone-stimulated cAMP formation were mediated by the AT1 receptor subtype. In bovine adrenals, only AT1 receptors were detected in membranes prepared from the cortex and the medulla, as well as in cultured glomerulosa cells. The relative inhibitory potency of Dup 753 was lower by an order of magnitude at bovine than at rat AT1 receptors. The inhibition of AII-induced aldosterone production by the various antagonists was closely correlated with their inhibitory potencies on 125I-[Sar1,Ile8]AII binding to bovine glomerulosa cells. These data suggest that the known effects of AII in adrenal glomerulosa cells are mediated through the AT1 receptor subtype and that the distribution and/or specificity of the AT2 receptors shows marked species variations.

Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry.

The relationships between receptor-mediated endocytosis and the generation of intracellular signals were analyzed in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In cells equilibrated with 125I-AII analogs at 4 degrees C, specifically bound agonist but not antagonist AII derivatives were rapidly internalized at 37 degrees C. AII-induced internalization was not influenced by the presence or absence of extracellular Ca2+ but was inhibited by treatment with phenylarsine oxide (PAO) or by arresting coated pit formation with hypotonic shock and potassium depletion. Inhibition of internalization by PAO was prevented by the bifunctional sulfhydryl reagent dithiothreitol but only partially reversed by mercaptoethanol, and readdition of K+ restored internalization in K(+)-depleted cells. Treatment with PAO did not impair the initial AII-induced elevations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and cytoplasmic calcium [( Ca2+]i) but reduced the sustained phase of the Ins(1,4,5)P3 response by 85% and abolished the second phase of the cytoplasmic Ca2+ response; these responses were restored by concomitant treatment with dithiothreitol. Inhibition of AII-receptor internalization by K+ depletion also caused selective loss of the sustained phase of the AII-induced Ca2+ response. Thus, blockade of AII-receptor internalization has similar effects as extracellular Ca2+ deficiency, which abolishes the sustained but not the early AII-induced increases in Ins(1,4,5)P3 production and [Ca2+]i. The close correlations between AII-induced internalization and the generation of Ins(1,4,5)P3 and [Ca2+]i responses suggest that endocytosis of the agonist-receptor complex is necessary to maintain the production of these intracellular signals. It is also possible that receptor-operated vesicular uptake of extracellular Ca2+ makes a significant contribution to the sustained [Ca2+]i responses of certain agonist-stimulated target cells.

Modulation of agonist-induced inositol phosphate metabolism by cyclic adenosine 3',5'-monophosphate in adrenal glomerulosa cells.

Activation of the cAMP messenger system was found to cause specific changes in angiotensin-II (All)-induced inositol phosphate production and metabolism in bovine adrenal glomerulosa cells. Pretreatment of [3H]inositol-labeled glomerulosa cells with 8-bromo-cAMP (8Br-cAMP) caused both short and long term changes in the inositol phosphate response to stimulation by All. Exposure to 8Br-cAMP initially caused dose-dependent enhancement (ED50 = 0.7 microM) of the stimulatory action of All (50 nM; 10 min) on the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its immediate metabolites. This effect of 8Br-cAMP was also observed in permeabilized [3H]inositol-labeled glomerulosa cells in which degradation of Ins(1,4,5)P3 was inhibited, consistent with increased activity of phospholipase-C. Continued exposure to 8Br-cAMP for 5-16 h caused selective enhancement of the All-induced increases in D-myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4] and myo-inositol 1,4,5,6-tetrakisphosphate. The long term effect of 8Br-cAMP on the 6-phosphorylated InsP4 isomers, but not the initial enhancement of Ins(1,4,5)P3 formation, was inhibited by cycloheximide. The characteristic biphasic kinetics of All-induced Ins(1,4,5)P3 formation were also changed by prolonged treatment with 8Br-cAMP to a monophasic response in which Ins(1,4,5)P3 increased rapidly and remained elevated during All stimulation. In permeabilized glomerulosa cells treated with 8Br-cAMP for 16 h, the conversion of D-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] to Ins(1,3,4,6)P4 was consistently increased, whereas dephosphorylation of Ins(1,4,5)P3 to D-myo-inositol 1,4-bisphosphate and of D-myo-inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4)P3, was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor.

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.

Regulation of 1,2-diacylglycerol production by angiotensin-II in bovine adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin-II (AII) induced a biphasic increase in 1,2-sn-diacylglycerol (DAG), with an initial peak at 10 sec followed by a transient decrease at 30 sec. The second increase was much higher in magnitude than the first peak and reached its maximum after 1 h of stimulation. Such kinetics of DAG formation resemble those with which AII stimulates the formation of inositol-1,4,5-trisphosphate. The protein synthesis inhibitor cycloheximide, which prevents hormone-induced de novo phospholipid synthesis in adrenal fasciculata cells, had no effect on the DAG response to Aii. The first phase of signal generation of both inositol-1,4,5-trisphosphate and DAG was not affected by incubation in calcium-deficient extracellular medium. However, the second phase of the inositol phosphate response was almost completely inhibited in low calcium medium, while the DAG response was reduced by only one third. Pertussis toxin (150 ng/ml) and the voltage-sensitive calcium channel inhibitors, nifedipine (1 microM) and Ni2+ (100 microM), had no effect on the DAG response to AII. The retention of a substantial DAG response to AII in low calcium medium, with concomitant diminution of the inositol phosphate response, indicates that a major part of the DAG formed during the sustained phase of hormonal stimulation is derived from sources other than phosphoinositides. The DAGs produced from different phospholipids could have distinctive fatty acid compositions and membrane localizations, which, in turn, could result in the differential activation of protein kinase-C. In this way, the increased complexity of the hormonally induced signalling pathway could allow for a greater diversity of responses in hormone-stimulated target cells.

Metabolism of inositol pentakisphosphate to inositol hexakisphosphate in Xenopus laevis oocytes.

The formation and metabolism of inositol pentakis-and hexakisphosphates (InsP5 and InsP6) were investigated in Xenopus laevis oocytes. After [3H]inositol injection, [3H]InsP5 and subsequently [3H]Insp6 increased progressively over 72 h. In intact oocytes, [3H]InsP5 was progressively converted to [3H]InsP6 from 6 to 72 h of incubation and was not metabolized to lower inositol phosphates. In contrast, [3H]InsP6 remained unmetabolized for up to 72 h. These data are consistent with the kinetics of the increases in [3H]InsP5 and [3H]InsP6 in [3H]inositol-labeled oocytes. The highly phosphorylated inositols showed significant changes during oogenesis and maturation. In oocytes incubated for 48 h after [3H]inositol injection, the radioactive incorporation into polyphosphoinositols increased progressively from stage 3 to stage 6, with 5- and 6-fold rises (cpm/mg protein) for [3H]InsP5 and [3H]InsP6, respectively. These developmental changes were associated with 5-fold increases in [3H]inositol tetrakisphosphate between stages 3 and 6 of oogenesis. Induction of oocyte maturation by progesterone (1 microM) during the last 12 of a 36-h incubation with [3H]inositol doubled the levels of [3H]InsP6 relative to [3H]InsP5, suggesting that the activity of inositol pentakisphosphate kinase increases during maturation. These results provide direct evidence for metabolic conversion of InsP5 to InsP6 in animal cells and show that the higher inositol polyphosphates, unlike the lower phosphoinositols, are extraordinarily stable. These species increase markedly during ovum development and may play a regulatory role in oogenesis and maturation.

Agonist-induced regulation of inositol tetrakisphosphate isomers and inositol pentakisphosphate in adrenal glomerulosa cells.

In adrenal glomerulosa cells, angiotensin II (AII) rapidly stimulates the formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and causes marked long-term changes in the levels of highly phosphorylated inositols. Glomerulosa cells prelabeled with [3H]inositol for 48 h and exposed to AII for 10 min showed prominent increases in inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and smaller increases in two additional tetrakisphosphates, Ins-1,3,4,6-P4 and another (Ins-3,4,5,6-P4) eluting in the position of Ins-3,4,5,6-P4 and its stereoisomer, Ins-1,4,5,6-P4, on anion exchange liquid chromatography. A concomitant decrease in InsP5 indicates that an increase in Ins-1,4,5,6-P4, the breakdown product of InsP5, is probably responsible for the initial rise in Ins-3,4,5,6-P4 during 10 min stimulation by AII. During prolonged stimulation by AII, Ins-1,3,4,5-P4 began to decline from its high, stimulated level after the first hour but the level of Ins-1,3,4,6-P4 remained elevated for several hours. There were also progressive increases in the levels of Ins-3,4,5,6-P4 and InsP5 during stimulation for up to 16 h with AII. Treatment of adrenal cells for 16 h with the cyclic AMP-mediated secretagogue, adrenocorticotropic hormone (ACTH), slightly increased basal levels of Ins-1,3,4,6-P4, Ins-3,4,5,6-P4, and InsP5, and enhanced the subsequent AII-stimulated increases in the two additional tetrakisphosphate isomers but not of inositol trisphosphates or Ins-1,3,4,5-P4. This change in the pattern of the higher inositol phosphate response to AII was manifested within 2 h after exposure to ACTH, and was mimicked by treatment with 8-bromo cyclic AMP or forskolin. Treatment with 50 microM cycloheximide abolished the ACTH-induced increases in inositol polyphosphate responses during AII stimulation, but had no effect on the responses of untreated cells to AII. The conversion of [3H]Ins-1,3,4-P3 to [3H]Ins-1,3,4,6-P4, a reaction linking the receptor-mediated InsP3 response to higher inositol phosphates, was enhanced in permeabilized cells that were pretreated for 16 h with either ACTH or AII. These results demonstrate that the reactions by which Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4 are formed and converted to InsP5 are influenced by agonist-stimulated regulatory processes that include both calcium-dependent and cyclic AMP-dependent mechanisms of target cell activation. They also reveal changes consistent with agonist-induced conversion of InsP5 to its dephosphorylated metabolite, Ins-1,4,5,6-P4, during short-term stimulation by AII.

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells.

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.

Metabolism of inositol 1,4,5-trisphosphate to higher inositol phosphates in bovine adrenal cytosol.

The metabolism of inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate was studied in a cytosolic fraction prepared from the bovine adrenal cortex. The activity of the partially purified inositol 1,4,5-trisphosphate 3-kinase was dependent on Ca2+/calmodulin, Mg2+, and pH, and was inhibited by 2,3-bisphosphoglycerate. The enzyme exhibited Michaelis-Menten behavior toward its two substrates, inositol 1,4,5-trisphosphate and ATP, with Km values of 0.42 mumol/L and 0.4 mmol/L, respectively. The presence of other inositol-phosphate metabolizing enzymes in the cytosolic fraction was indicated by the appearance of additional inositol polyphosphates during prolonged incubation with inositol 1,4,5-trisphosphate. These included inositol 1,3,4-trisphosphate, inositol 1,3,4,6-tetrakisphosphate, and inositol pentakisphosphate. These findings are consistent with the rapid phosphorylation of inositol 1,4,5-trisphosphate to the 1,3,4,5-tetrakisphosphate by the calcium/calmodulin-dependent 3-kinase, and its subsequent conversion to inositol 1,3,4-trisphosphate and thence to inositol 1,3,4,6-tetrakisphosphate in angiotensin-stimulated bovine glomerulosa cells. The formation of inositol pentakisphosphate during prolonged incubations suggests that inositol 1,3,4,6-tetrakisphosphate is slowly phosphorylated and serves as a source of inositol pentakisphosphate in the adrenal. The metabolic conversion of inositol 1,4,5-trisphosphate to several higher inositol polyphosphates provides potential new messengers for intracellular regulation in agonist-stimulated target cells.

Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II.

Stimulation of aldosterone production by angiotensin II in the adrenal glomerulosa cell is mediated by increased phosphoinositide turnover and elevation of intracellular Ca2+ concentration. In cultured bovine glomerulosa cells, angiotensin II caused rapid increases in inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) levels and cytosolic Ca2+ during the first minute of stimulation, when both responses peaked between 5 and 10 s and subsequently declined to above-baseline levels. In addition to this temporal correlation, the dose-response relationships of the angiotensin-induced peak increases in cytosolic Ca2+ concentrations and Ins-1,4,5-P3 levels measured at 10 s were closely similar. However, at later times (greater than 1 min) there was a secondary elevation of Ins-1,4,5-P3, paralleled by increased formation of inositol 1,3,4,5-tetrakisphosphate that was associated with cytosolic Ca2+ concentrations only slightly above the resting value. These results are consistent with the primary role of Ins-1,4,5-P3 in calcium mobilization during activation of the glomerulosa cell by angiotensin II. They also suggest that Ins-1,4,5-P3 participates in the later phase of the target-cell response, possibly by acting alone or in conjunction with its phosphorylated metabolites to promote calcium entry and elevation of cytosolic Ca2+ during the sustained phase of aldosterone secretion.