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Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1Aangiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220Mdecreased agonist-induced receptor phosphorylation by ∼40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by ∼30%. The inhibitory effects of GRK2K220Mexpression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1receptor.

The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is beta-arrestin- and dynamin-dependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a beta-arrestin- and dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of beta-arrestin1(V53D) and beta-arrestin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT(1) receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of beta-arrestin- and dynamin-dependent endocytosis of the AT1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations.

The angiotensin AT(2) receptor is an atypical seven transmembrane domain receptor that is coupled to activation of tyrosine phosphatase and inhibition of MAP kinase, and does not undergo agonist-induced internalization. An investigation of the occurrence and nature of AT(2) receptor phosphorylation revealed that phorbol ester-induced activation of protein kinase C (PKC) in HA-AT(2) receptor-expressing COS-7 cells caused rapid and specific phosphorylation of a single residue (Ser(354)) located in the cytoplasmic tail of the receptor. Agonist activation of AT(2) receptors by angiotensin II (Ang II) also caused rapid PKC-dependent phosphorylation of Ser(354) that was prevented by the AT(2) antagonist, PD123177, and by inhibitors of PKC. In cells coexpressing AT(1) and AT(2) receptors, Ang II-induced phosphorylation of the AT(2) receptor was reduced by either PD123177 or the AT(1) receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT(2) receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation of the AT(1) receptor. The latter process may regulate the counteracting effects of AT(2) receptors on growth responses to AT(1) receptor activation.

The C-terminal region of the third intracellular loop of the AT1angiotensin receptor (AT1-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of Gq/11-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232–240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile238or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile238with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe239with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile238and Phe239mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile238and Phe239as the critical residues in the C-terminal region of the third intracellular loop of the AT1-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile238of the AT1-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.

Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution. TheSchizosaccharomyces pombe sod2gene provides a useful model system to analyze this process. sod2is near the telomere of chromosome I and encodes a plasma membrane Na+(Li+)/H+antiporter. Whensod2is amplified, S. pombesurvives otherwise lethal concentrations of LiCl, and >90% of the amplifiedsod2genes are found in 180- and 225-kilobase (kb) linear amplicons. The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II. The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I. The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer. LiCl-resistant mutants arise 200–600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons. These data suggest that amplicon formation may begin with DNA lesions such as breaks. In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon.

Gene amplification in eukaryotes plays an important role in drug resistance, tumorigenesis, and evolution. The Schizosaccharomyces pombe sod2 gene provides a useful model system to analyze this process. sod2 is near the telomere of chromosome I and encodes a plasma membrane Na(+)(Li(+))/H(+) antiporter. When sod2 is amplified, S. pombe survives otherwise lethal concentrations of LiCl, and >90% of the amplified sod2 genes are found in 180- and 225-kilobase (kb) linear amplicons. The sequence of the novel joint of the 180-kb amplicon indicates that it is formed by recombination between homologous regions near the telomeres of the long arm of chromosome I and the short arm of chromosome II. The 225-kb amplicon, isolated three times more frequently than the 180-kb amplicon, is a palindrome derived from a region near the telomere of chromosome I. The center of symmetry of this palindrome contains an inverted repeat consisting of two identical 134-base pair sequences separated by a 290-base pair spacer. LiCl-resistant mutants arise 200-600 times more frequently in strains deficient for topoisomerases or DNA ligase activity than in wild-type strains, but the mutant cells contain the same amplicons. These data suggest that amplicon formation may begin with DNA lesions such as breaks. In the case of the 225-kb amplicon, the breaks may lead to a hairpin structure, which is then replicated to form a double-stranded linear amplicon, or to a cruciform structure, which is then resolved to yield the same amplicon.

When the electric field below a thunderstorm or other electrified cloud is around 10 kV/m, it is sometimes possible to initiate (“trigger”) an upward‐propagating lightning‐leader by launching a rocket that uncoils a wire from the ground. The triggered leader propagates upward from the tip of the wire lifted by the rocket. When the channel is hot enough, a flash is visible. Triggering is common when the leader carries positive charge, but not when it carries negative charge. This article is about four flashes consisting of triggered negative leaders that branched into low‐altitude regions of positive cloud charge over Langmuir Laboratory in central New Mexico. Measurements of current and the locations of leader channels are available for three of the four flashes. Some current pulses at the ground for Flash 2 originated at negative leader steps more than 3 km away, which is a greater distance than has been reported from video measurements. Flashes 3 and 4 propagated only into thunderstorm lower positive charge, and the average lightning‐charge densities inside the volumes occupied by these two flashes are remarkably close. Our best estimate of density for Flashes 3 and 4 lies between −4.2 and −1.8 C/km3, which is compatible with the large spread in cloud‐charge densities derived from instruments carried on airplanes or balloons into low positive regions in thunderstorms. Triggered negative leader boundaries lie within low positive cloud boundariesLightning‐charge densities (−4.2 to −1.8 C/km3) are within the range of cloud‐charge densities deduced from airborne measurementsStep recoil waves propagate farther than 3 km backward from negative leader steps Triggered negative leader boundaries lie within low positive cloud boundaries Lightning‐charge densities (−4.2 to −1.8 C/km3) are within the range of cloud‐charge densities deduced from airborne measurements Step recoil waves propagate farther than 3 km backward from negative leader steps

Sodium uptake by rat adrenal glomerulosa cells was stimulated by intracellular acidosis evoked by Na+-propionate. This process was inhibited by 5-(N,N-hexamethylene) amiloride (HMA), a known inhibitor of the Na+-H+ exchange. These experiments demonstrate the existence of the Na+-H+ exchange in glomerulosa cells. Although amiloride inhibited the angiotensin II- and adrenocorticotropic hormone (ACTH)-induced aldosterone response, HMA, a more specific inhibitor of Na+-H+ exchange, failed to do that. 45Ca2+ influx and efflux were dependent on intra- and extracellular Na+ concentrations. Amiloride analogues, known to inhibit Na+-Ca2+ exchange, reduced basal 45Ca influx. Although we could not reveal the activation of Na+-Ca2+ exchange by angiotensin II, inhibitors of Na+-Ca2+ exchange also inhibited the angiotensin- and ACTH-induced aldosterone response of glomerulosa cells. Our results suggest that Na+-Ca2+ exchange supports the maintenance of basal Ca2+ level in the cytoplasma of glomerulosa cells, and amiloride derivatives inhibit aldosterone production by reducing Ca2+ level below resting values.

The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.

For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1, Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.

Recently it has been suggested [(1987) Nature 325, 456–458; (1987) FEBS Lett. 212, 123–126] that the activation of Na+/H+exchange is a prerequisite for platelet aggregation and the development of the Ca2+signal. As direct evidence for the role of the Na+/H+‐exchange pathway the inhibition of the Ca2+signal by EIPA, a specific inhibitor of Na+/H+exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 μM) completely block Na+/H+exchange while EIPA inhibits aggregation or Ca2+mobilization only in concentrations 100‐times greater than 1 μM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+exchange, does not affect Na+/H+exchange in platelets but blocks aggregation and Ca2+mobilization. We conclude that while Na+/H+exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+signal and aggregation.

Turnover of 32P‐labelled phosphatidylinositol (PI) was examined in isolated adrenal glomerulosa cells. Increased incorporation of [32P] phosphate into PI in response to angiotensin II was completely prevented by Li+. A simultaneous accumulation of 32P activity in phosphatidic acid (PA) was also observed. Angiotensin II increased the breakdown of PI despite the presence of Li+. These results suggest that Li is a suitable tool to interrupt the accelerated PI cycle in angiotensin‐stimulated cells. Aldosterone production of superfused cells was inhibited by Li+when the cells were stimulated with angiotensin II. On the other hand, Li+did not inhibit the aldosterone response of the cells to ACTH, a hormone which acts via cyclic AMP and does not enhance PI turnover in these cells. On the basis of these results, we assume that the inhibitory effect of Li+on aldosterone production is related to its effect on PI turnover.

Angiotensin II (AII) interacts with specific receptors in the adrenal glomerulosa cell and stimulates the hydrolysis of plasma membrane phosphoinositides by phospholipase C, with production of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and subsequent mobilization of intracellular Ca2+. In electrically permeabilized, [3H]inositol-labeled glomerulosa cells, AII stimulated Ins-1,4,5-P3 production within 15 s with half-maximal potency of 10(-9) M. The nonhydrolyzable GTP analog, guanosine 5'-O-thiotriphosphate (GTP gamma S), stimulated Ins-1,4,5-P3 formation in a dose-dependent manner with half-maximal effect at 10(-7) M. AII-activated Ins-1,4,5-P3 production was further increased by guanine nucleotides. The rate at which GTP gamma S-stimulated inositol polyphosphate production was consistently slower than that of AII. In adrenal membrane preparations, GTP gamma S-stimulated polyphosphoinositide hydrolysis was enhanced by Ca2+, with half-maximal activity at 300 nM free Ca2+. Ins-1,4,5-P3 formation was also increased by NaF, further indicating the involvement of a guanine nucleotide regulatory protein. In addition to Ins-1,4,5-P3 and its metabolites formed during degradation via the 4-monophosphate pathway, AII and GTP gamma S stimulated the formation of the phosphorylated metabolite inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in permeabilized cells. The absence of a significant rise in inositol 1-monophosphate indicated that phosphatidylinositol hydrolysis was not stimulated by AII or GTP gamma S. Pretreatment of glomerulosa cells with pertussis toxin for 12 h before permeabilization did not inhibit AII- or GTP gamma S-stimulated inositol polyphosphate formation. However, treatment with cholera toxin, forskolin, or 8-Br-cAMP for 12 h enhanced both basal and ligand-stimulated Ins-1,4,5-P3 production. These observations suggest that agonist binding to the AII receptor activates a polyphosphoinositide-specific phospholipase C in the adrenal glomerulosa cell, and that a distinctive guanine regulatory protein is involved in this mechanism.

A series of deletion mutants was created to analyze the function of the membrane‐proximal region of the cytoplasmic tail of the rat type 1a (AT1a) angiotensin receptor. In transiently transfected COS‐7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar1,Ile8]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II‐induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine309and adjacent lysine residues as significant determinants of AT1areceptor expression.

The Ca2+ signal induced by an increase in extracellular K+ concentration from 3.6 to 5.6 mM or angiotensin II (ANG II) was inhibited by the dihydropyridine (DHP) Ca2+ channel blocker, nifedipine, and enhanced by the DHP Ca2+ channel agonist, BAY K 8644. The DHP sensitivity of the ANG II-induced Ca2+ response was already detectable during the peak phase, suggesting that the DHP receptor plays an important role during the initial phase of ANG II stimulation. K+ and ANG II stimulated a nifedipine-sensitive Mn2+ influx pathway, further promoting the role of a DHP receptor in their mechanism of action. Fluorescent membrane potential measurements showed that, in contrast to the rapid depolarization induced by K+, the ANG II-induced depolarization had a lag time of > 30 s. The slow kinetics of depolarization compared with the immediate effect of ANG II on Mn2+ influx and the DHP sensitivity of the initial Ca2+ peak indicates that ANG II initiates the activation of the DHP-sensitive Ca2+ channel by a mechanism other than depolarization.

We examined the effect of the depletion of intracellular Ca2+ stores on Ca2+ influx in rat glomerulosa cells. Depletion of intracellular Ca2+ stores was achieved by inhibiting sarco/endoplasmic reticulumtype Ca(2+)-ATPase with thapsigargin or 2,5,di-(t-butyl)-1,4-benzohydroquinone (t-BHQ). Both inhibitors induced a sustained rise in cytoplasmic Ca2+ concentration. The initial rise was observed also in Ca(2+)-free medium, while the sustained phase disappeared, indicating that the latter requires Ca2+ influx. In Ca(2+)-free medium, the readdition of Ca2+ induced a steeper and higher rise in intracellular Ca2+ concentration in thapsigargin-treated cells than in controls, supporting the role of Ca2+ influx. In normal medium, the addition of Cd2+ (80 microM) evoked an immediate inhibition of the sustained phase of thapsigargin response. The response to thapsigargin was insensitive to nifedipine. Thapsigargin failed to enhance Mn2+ quenching of fura 2. Our results provide evidence for the existence of capacitative Ca2+ influx in rat glomerulosa cells and indicate that dihydropyridine-sensitive Ca2+ channels do not participate in capacitative Ca2+ entry. High concentrations of thapsigargin and t-BHQ, similar to the reported effects of angiotensin II and vasopressin, inhibited K(+)-induced Ca2+ signals. These effects appear, however, to be independent of the depletion of internal Ca2+ stores.

Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration.

The type 1 angiotensin II (AT1) receptor undergoes rapid endocytosis and down-regulation after agonist binding. In studies on the structural determinants of agonist-induced endocytosis, serial deletions in the cytoplasmic tail of the rat AT1a receptor showed that the carboxyl-terminal 22 amino acids are not necessary for its internalization. However, internalization was markedly impaired by the removal of one additional amino acid (Leu337) and was reduced by 95% after removal of Ser335 and Thr336. Single alanine replacements of amino acids in this region showed that individual substitutions of Thr332, Ser335, Thr336, Leu337, and Ser338 caused moderate but significant impairment of the internalization rate. Replacement of both Ser335 and Thr336 with alanine residues further impaired the internalization rate, and triple alanine replacement of the Ser-Thr-Leu motif reduced internalization to almost the same extent as the corresponding tail deletion mutant. The Ser-Thr-Leu motif is highly conserved in mammalian AT1 receptors but is not present in the noninternalizing type 2 angiotensin II receptor. These data demonstrate that a serine/threonine-rich region including Leu337 in the cytoplasmic tail of the AT1 receptor is a major requirement for endocytosis of the hormone-receptor complex and support the concept that similar motifs in other G protein-coupled receptors are determinants of their agonist-induced internalization.

Initial 45Ca uptake was measured in isolated rat glomerulosa cells. A small reduction in membrane potential produced by increasing the K+concentration from 2 to 3·6 mmol/l stimulated 45Ca uptake by about 35%, while a bigger depolarization induced by 18·5 mmol K+/l increased the uptake by about 100%. Since Ca2+influx was already activated at a calculated membrane potential below −70 mV, and was found to be sensitive to the dihydropyridine antagonist nifedipine (1 μmol/l), but insensitive to nickel ions (100 μmol/l), it does not meet the criteria established for T- or L-type voltage-dependent Ca2+channels. Exposure of glomerulosa cells to angiotensin II (AII) for 10 min also enhanced the rate of 45Ca influx. The effect of AII was not sensitive to 1 μmol nifedipine/l, but was strongly inhibited by 5-(N-4-chlorobenzyl)-N-(2′,4′-dimethyl)benzamil (CBDMB, 30 μmol/l), an inhibitor of the Na+/Ca2+antiporter. These observations suggest that during the sustained phase of stimulation with AII, a CBDMB-sensitive mechanism, rather than dihydropyridine-sensitive calcium channels, is involved in Ca2+uptake in rat glomerulosa cells. The bulk Ca2+influx did not correlate with aldosterone production; however, the maintained activity of different Ca2+entry mechanisms seems to be essential for AII-induced aldosterone production.Journal of Endocrinology(1989) 122,361–370

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 InsP4isomers was previously identified as Ins-1,3,4,6-P4and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4isomer, 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-P4or Ins-3,4,5,6-P4(= L-Ins-1,4,5,6-P4) for the third InsP4isomer. 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-P4and 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-P4and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5in permeabilized bovine glomerulosa cells. In addition, InsP5itself 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 InsP3response by the conversion of Ins-1,3,4-P3through Ins-1,3,4,6-P4to 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.

Recently it has been suggested [(1987) Nature 325, 456–458; (1987) FEBS Lett. 212, 123–126] that the activation of Na +/H +exchange is a prerequisite for platelet aggregation and the development of the Ca 2+signal. As direct evidence for the role of the Na +/H +-exchange pathway the inhibition of the Ca 2+signal by EIPA, a specific inhibitor of Na +/H +exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 μM) completely block Na +/H +exchange while EIPA inhibits aggregation or Ca 2+mobilization only in concentrations 100-times greater than 1 μM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na +/Ca 2+exchange, does not affect Na +/H +exchange in platelets but blocks aggregation and Ca 2+mobilization. We conclude that while Na +/H +exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca 2+signal and aggregation.

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 Asn298and was reduced by replacement of Pro299or 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 Phe301residue contributes significantly to agonist binding, and Asn298is required for normal receptor activation and signal transduction.

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.

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function. (Gastroenterology 1997 Jun;112(6):1948-60)

The rat type 1a (AT1a) angiotensin II (Ang II) receptor contains a highly conserved tyrosine residue in the fifth transmembrane region that is present in most G protein-coupled receptors. The role of this amino acid in AT1 receptor activation was analyzed in a mutant receptor (Y215F) created by replacing Tyr215 with phenylalanine. The mutant receptor was highly expressed in transfected COS-7 cells, and its binding affinity for the peptide antagonist [Sar1,Ile8]Ang II was similar to that of the wild type receptor. Although the structural integrity of the peptide ligand binding domain was preserved in the Y215F mutant receptor, its affinity for the native agonist, Ang II, was significantly reduced. Also, whereas guanosine 5'-3-O-(thio)triphosphate markedly reduced Ang II binding to the wild type receptor, it had little effect on agonist binding to the mutant receptor. Agonist-induced internalization of the mutant receptor was also impaired, and its ability to mediate inositol phosphate responses to Ang II stimulation was abolished. The concomitant decreases in receptor internalization and G protein-mediated signaling of the Y215F mutant receptor indicate that Tyr215 has a critical role in AT1 receptor activation. In view of its conservation among members of the seven transmembrane domain receptor superfamily, this residue is likely to be of general importance in signal transduction from G protein-coupled receptors.

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.

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-P4and another (Ins-3,4,5,6-P4) eluting in the position of Ins-3,4,5,6-P4and 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-P4during 10 min stimulation by AII. During prolonged stimulation by AII, Ins-1,3,4,5-P4began to decline from its high, stimulated level after the first hour but the level of Ins-1,3,4,6-P4remained elevated for several hours. There were also progressive increases in the levels of Ins-3,4,5,6-P4and 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 εMcycloheximide 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-P3to [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-P4and Ins-3,4,5,6-P4are formed and converted to InsP5are 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 InsP5to its dephosphorylated metabolite, Ins-1,4,5,6-P4, during short-term stimulation by AII.