scholarly journals Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+

1997 ◽  
Vol 328 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Thomas J. A. CARDY ◽  
David TRAYNOR ◽  
W. Colin TAYLOR
Keyword(s):  
Inositol Trisphosphate ◽  
Receptor Subtype ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Single Mechanism ◽  
Receptor Isoforms ◽  
Type 3

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mobilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of each receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the endogenous receptors. The expressed receptors were glycosylated and assembled into tetramers, and binding of [3H]IP3 to each subtype was regulated by cytosolic Ca2+. The effects of increased [Ca2+] on native cerebellar and type-1 receptors expressed in Sf9 cells were indistinguishable. A maximally effective increase in [Ca2+] reversibly inhibited [3H]IP3 binding by approx. 50% by decreasing the number of IP3-binding sites (Bmax) without affecting their affinity for IP3. The effects of Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [3H]IP3 binding by increasing Bmax, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors although by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differences is that, at resting cytosolic [Ca2+], type-3 receptors are more sensitive than type-1 receptors to IP3, but the situation reverses at higher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evoked by receptors linked to IP3 formation.

Selective recognition of inositol phosphates by subtypes of the inositol trisphosphate receptor

2001 ◽  
Vol 355 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V. L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Hydroxy Group ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Subtype Selectivity ◽  
Type 3

Synthetic analogues of inositol trisphosphate (IP3), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP3, were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP3 receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [3H](1,4,5)IP3 in Ca2+-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP3 were type 3 (Kd = 11±2nM)>type 2 (Kd = 17±2nM) > type 1 (Kd = 24±4nM). (1,4,5)IP3 binding was reversibly stimulated by increased pH, but the subtypes differed in their sensitivity to pH (type 1 > type 2>type 3). For all three subtypes, the equatorial 6-hydroxy group of (1,4,5)IP3 was essential for high-affinity binding, the equatorial 3-hydroxy group significantly improved affinity, and the axial 2-hydroxy group was insignificant; a 1-phosphate (or in its absence, a 2-phosphate) improved binding affinity. The subtypes differed in the extents to which they tolerated inversion of the 3-hydroxy group of (1,4,5)IP3 (type 1>type 2>type 3), and this probably accounts for the selectivity of (1,4,6)IP3 for type 1 receptors. They also differed in their tolerance of inversion, removal or substitution (by phosphate) of the 2-hydroxy group (types 2 and 3>type 1), hence the selectivity of (1,2,4,5)IP4 for type 2 and 3 receptors. Removal of the 3-hydroxy group or its replacement by fluorine or CH2OH was best tolerated by type 3 receptors, and accounts for the selectivity of 3-deoxy(1,4,5)IP3 for type 3 receptors. Our results provide the first systematic analysis of the recognition properties of IP3 receptor subtypes and have identified the 2- and 3-positions of (1,4,5)IP3 as key determinants of subtype selectivity.

Functional properties of Drosophila inositol trisphosphate receptors

2001 ◽  
Vol 359 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Jane E. SWATTON ◽  
Stephen A. MORRIS ◽  
Frank WISSING ◽  
Colin W. TAYLOR
Keyword(s):  
Functional Properties ◽  
Inositol Trisphosphate ◽  
Stable State ◽  
Hill Coefficient ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
S2 Cells ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Adenophostin A

The functional properties of the only inositol trisphosphate (IP3) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP3 receptors of S2 cells bound (1,4,5)IP3 with high affinity (Kd = 8.5±1.1nM), mediated positively co-operative Ca2+ release from a thapsigargin-sensitive Ca2+ store (EC50 = 75±4nM, Hill coefficient = 2.1±0.2), and they were recognized by an antiserum to a peptide conserved in all IP3 receptor subtypes in the same way as mammalian IP3 receptors. As with mammalian IP3 receptors, (2,4,5)IP3 (EC50 = 2.3±0.3μM) and (4,5)IP2 (EC50 approx. 10μM) were approx. 20- and 100-fold less potent than (1,4,5)IP3. Adenophostin A, which is typically approx. 10-fold more potent than IP3 at mammalian IP3 receptors, was 46-fold more potent than IP3 in S2 cells (EC50 = 1.67±0.07nM). Responses to submaximal concentrations of IP3 were quantal and IP3-evoked Ca2+ release was biphasically regulated by cytosolic Ca2+. Using rapid superfusion to examine the kinetics of IP3-evoked Ca2+ release from S2 cells, we established that IP3 (10μM) maximally activated Drosophila IP3 receptors within 400ms. The activity of the receptors then slowly decayed (t1/2 = 2.03±0.07s) to a stable state which had 47±1% of the activity of the maximally active state. We conclude that the single subtype of IP3 receptor expressed in Drosophila has similar functional properties to mammalian IP3 receptors and that analyses of IP3 receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP3 receptors.

scholarly journals Determinants of adenophostin A binding to inositol trisphosphate receptors

2002 ◽  
Vol 367 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Stephen A. MORRIS ◽  
Edmund P. NEROU ◽  
Andrew M. RILEY ◽  
Barry V.L. POTTER ◽  
Colin W. TAYLOR
Keyword(s):  
Binding Domain ◽  
Sf9 Cells ◽  
Receptor Subtypes ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
High Affinity ◽  
Recombinant Type ◽  
Adenophostin A ◽  
Binding Core

Inositol 1,4,5-trisphosphate (IP3) receptors from cerebellum and recombinant type 1 IP3 receptors expressed in Sf9 cells had indistinguishable affinities for IP3 (Kd = 6.40±0.48nM) and adenophostin A (Kd = 0.89±0.05nM). In cytosol-like medium, each of the three mammalian IP3 receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP3. It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [3H]IP3 from type 1 IP3 receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [3H]IP3 binding to the 1—604 protein, but abolished binding to the 224—604 protein. The 1—604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP3, suggesting that C-terminal residues selectively inhibit IP3 binding. The 224—604S1+ fragment bound IP3 and adenophostin A with increased affinity, but as with the 1—604 fragment it bound adenophostin A with only 2-fold greater affinity than IP3. High-affinity binding of adenophostin A may be partially determined by its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with residues within the IP3-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP3 for the minimal IP3-binding domain. In addition we suggest that C-terminal residues, which impede access of IP3, may selectively interact with adenophostin A to allow it unhindered access to the IP3-binding domain.

scholarly journals Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization

1999 ◽  
Vol 344 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Jane E. SWATTON ◽  
Stephen A. MORRIS ◽  
Thomas J. A. CARDY ◽  
Colin W. TAYLOR
Keyword(s):  
Single Type ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rinm5f Cells ◽  
Intact Cells ◽  
Single Receptor ◽  
Insp3 Receptors ◽  
Type 3 ◽  
Insp3 Receptor

There are three subtypes of mammalian Ins(1,4,5)P3 (InsP3) receptor, each of which forms an intracellular Ca2+ channel. Biphasic regulation of InsP3 receptors by cytosolic Ca2+ is well documented in cells expressing predominantly type 1 or type 2 InsP3 receptors and might contribute to the regenerative recruitment of Ca2+ release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP3 receptors of RIN-5F cells, cells in which the InsP3 receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca2+ [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP3 receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP3 receptors in RINm5F cells. In unidirectional 45Ca2+ efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP3 released only a fraction of the InsP3-sensitive Ca2+ stores, indicating that responses to InsP3 are quantal. Increasing the cytosolic free [Ca2+] ([Ca2+]i) from approx. 4 to 186 nM increased the sensitivity of the Ca2+ stores to InsP3: the EC50 decreased from 281±15 to 82±2 nM. Further increases in [Ca2+]i massively decreased the sensitivity of the stores to InsP3, by almost 10-fold when [Ca2+]i was 2.4 μM, and by more than 3000-fold when it was 100 μM. The inhibition caused by 100 μM Ca2+ was fully reversed within 60 s of the restoration of [Ca2+]i to 186 nM. The effect of submaximal InsP3 concentrations on Ca2+ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca2+. We conclude that type 3 InsP3 receptors of RINm5F cells mediate quantal Ca2+ release and they are biphasically regulated by cytosolic Ca2+, either because a single type 1 subunit within the tetrameric receptor confers the Ca2+ inhibition or because the type 3 subtype is itself directly inhibited by Ca2+.

scholarly journals A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors

1998 ◽  
Vol 334 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Thomas J. A. CARDY ◽  
Colin W. TAYLOR
Keyword(s):  
Spodoptera Frugiperda ◽  
Inhibitory Effect ◽  
Ip3 Receptors ◽  
Inhibitory Effects ◽  
Independent Manner ◽  
S 100 ◽  
High Concentrations ◽  
Type 3 ◽  
Stimulation Of

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627–11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+–calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

scholarly journals Ca2+ and calmodulin differentially modulate myo-inositol 1,4,5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms

2000 ◽  
Vol 346 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Sara VANLINGEN ◽  
Henk SIPMA ◽  
Patrick DE SMET ◽  
Geert CALLEWAERT ◽  
Ludwig MISSIAEN ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Recombinant Proteins ◽  
Ip3 Receptor ◽  
Binding Characteristics ◽  
Binding Domains ◽  
Receptor Isoforms ◽  
Inositol 1,4,5 Trisphosphate ◽  
Adenophostin A ◽  
Trisphosphate Receptor

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.

Aliskiren, exendin-4, and insulin: their impact on endothelin receptor subtype(s) regulation/binding in type 1 diabetic rat hearts

2013 ◽  
Vol 91 (10) ◽  
pp. 830-838 ◽  
Author(s):  
Sawsan M. Al Lafi ◽  
Shushan B. Artinian ◽  
Suzan S. Boutary ◽  
Nadine S. Zwainy ◽  
Khalil M. Bitar ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Diabetic Rat ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Rat Hearts ◽  
Heart Perfusion ◽  
Glucagon Like Peptide 1 ◽  
Coronary Endothelium ◽  
The Impact

This study focuses on the impact of aliskiren and (or) glucagon-like peptide-1 analogue on the binding affinity/regulation of endothelin-1 (ET-1) to its receptor subtypes A (ETAR) and B (ETBR) at the level of the coronary endothelium and the cardiomyocytes in a type-1 diabetic rat model. Seven groups were used: (i) normal rats, (ii) rats with induced diabetes, (iii) rats with induced diabetes that were treated with insulin, (iv) rats with induced diabetes that were treated with exendin-4, (v) rats with induced diabetes that were treated with aliskiren, (vi) rats with induced diabetes that were co-treated with insulin plus aliskiren, and (vii) rats with induced diabetes that were co-treated with exendin-4 plus aliskiren. Heart perfusion with [125I]-ET-1 was employed to estimate ET-1 binding affinity (τ = 1/K–n) to ETAR and ETBR at the level of the coronary endothelium and the cardiomyocytes. Plasma ET-1 levels were measured using enzyme immunoassay, whereas densities of ETAR and ETBR were detected using Western blot. No significance differences were detected in the τ of ETAR and ETBR between normal and diabetic in cardiomyocytes and the coronary endothelium. Exendin-4 normalized the τ value for ETAR and ETBR on coronary endothelium, while aliskiren normalized it on cardiomyocytes. Furthermore, ETAR and ETBR densities were normalized with monotreatments of aliskiren and exendin-4, compared with up-regulated ETAR and down-regulated ETBR band densities in the diabetic animals. Our data indicate that aliskiren alleviates diabetes-associated hypertrophy in type 1 diabetes mellitus.

Expression of mRNAs for different types of IP3 receptors in rat kidneys

1995 ◽  
Vol 268 (6) ◽  
pp. F1046-F1052 ◽  
Author(s):  
T. Yang ◽  
Y. Terada ◽  
H. Nonoguchi ◽  
K. Tomita ◽  
F. Marumo
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Receptor Mrna ◽  
Collecting Ducts ◽  
Different Types ◽  
Medullary Collecting Duct

Cloning studies have extensively characterized two types of inositol 1,4,5-trisphosphate (IP3) receptors from the rat. An IP3 receptor from the cerebellum is referred to as type 1, and a second, recently described, receptor is referred to as the type 2 IP3 receptor. The significance of different types of IP3 receptors, especially in vivo in the kidney, is not fully understood. We investigated the localization of mRNAs encoding these two types of IP3 receptors in microdissected nephron segments of rats using reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Type 1 IP3 receptor mRNA displayed a widespread, although not uniform, distribution along the nephron. In contrast, type 2 IP3 receptor mRNA was confined almost exclusively to collecting ducts, suggesting specific expression of type 2 IP3 receptor in collecting ducts. We then detected mRNAs for the two types of IP3 receptors in collecting ducts in dehydrated rats. Dehydration downregulated type 2 IP3 receptor mRNA in cortical collecting duct, outer medullary collecting duct, and the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney. the initial part of inner medullary collecting duct (IMCD), but not in the terminal part of IMCD. It had no effect on type 1 IP3 receptor mRNA expression in collecting ducts. We propose that different types of IP3 receptors may have different functions in the rat kidney.

scholarly journals Intracellular Calcium Concentration in the Inositol Trisphosphate Receptor Type 1 Knockout Mouse

1999 ◽  
Vol 10 (10) ◽  
pp. 2094-2101
Author(s):  
MATSUHIKO HAYASHI ◽  
TOSHIAKI MONKAWA ◽  
TADASHI YOSHIDA ◽  
HIROYUKI SASAMURA ◽  
MINEO MATSUMOTO ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Reverse Transcription ◽  
Calcium Concentration ◽  
Hormone Receptors ◽  
Receptor Type ◽  
Wild Type ◽  
Ip3 Receptor ◽  
Reverse Transcription Pcr ◽  
Receptor Isoforms

Abstract. Recently, mice with a disrupted inositol trisphosphate (IP3) receptor type 1 allele were produced by gene targeting. To examine the role of IP3 receptor type 1 in the regulation of intracellular calcium concentration ([Ca2+]i) of glomerular cells, [Ca2+]i was measured with fura 2-acetoxymethyl-ester in the superfused glomeruli from homozygous and wild-type mice. [Ca2+]i was determined in calcium-free medium before and after the addition of 10-7 M endothelin-1 (ET-1) and 10-6 M angiotensin II (AngII). The expression of mRNA of IP3 receptor isoforms and hormone receptors in the glomeruli from these animals also was measured by quantitative reverse transcription-PCR with specific primers for IP3 receptor isoforms (types 1, 2, and 3), AngII receptor type 1, and ET receptors (types A and B). In homozygous mutants, the shorter mRNA of IP3 receptor type 1, which lacks the first exon, is transcribed. Basal [Ca2+]i and the responses to ET-1 and AngII in homozygous mutants (ET-1, 55 ± 7 nM to 73 ± 7 nM; AngII, 66 ± 6 to 91 ± 8 nM) were significantly lower than those in the wild-type mice (ET-1, 93 ± 13 nM to 162 ± 13 nM; AngII, 87 ± 7 to 147 ± 9 nM; P < 0.05 for both hormones) without significant changes in mRNA expression of hormone receptors. The results with quantitative reverse transcription-PCR also revealed that mRNA expression of the IP3 receptor gene family was not significantly different between the two groups. The present study clearly shows that IP3 receptor type 1 plays a major role in the regulation of [Ca2+]i in the glomeruli and that lack of an isoform of IP3 receptor in the glomeruli does not induce expression of the other isoforms of the IP3 receptor.

scholarly journals Expression of type 1 angiotensin II receptor subtypes and angiotensin II-induced calcium mobilization along the rat nephron.

1997 ◽  
Vol 8 (11) ◽  
pp. 1658-1667 ◽  
Author(s):  
N Bouby ◽  
A Hus-Citharel ◽  
J Marchetti ◽  
L Bankir ◽  
P Corvol ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Collecting Duct ◽  
At1 Receptor ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Angiotensin Ii Receptor ◽  
Nephron Segments

The localization of two type 1 angiotensin II receptor subtype mRNA, AT1A and AT1B, was determined by reverse transcription-PCR on microdissected glomeruli and nephron segments. The coupling sensitivity of these two receptor subtypes was evaluated by measuring variations in intracellular calcium ([Ca2+]i) elicited by angiotensin II (Ang II) in structures expressing either AT1A or AT1B mRNA, using Fura-2 fluorescence. The highest expression of AT1 mRNA was found in glomerulus, proximal tubule, and thick ascending limb. In glomerulus, AT1A and AT1B mRNA were similarly expressed, whereas in all nephron segments AT1A mRNA expression was dominant (approximately 84%). The increase in [Ca2+]i elicited by 10(-7) mol/L Ang II was highest in proximal segments (delta [Ca2+]i is approximately equivalent to 300 to 400 nmol/L) and thick ascending limb (delta [Ca2+]i is approximately equivalent to 200 nmol/L). In glomerulus and collecting duct, the response was lower (delta < 100 nmol/L). The median effective concentrations for Ang II were of the same order of magnitude in glomerulus (12.2 nmol/L), in which both AT1A and AT1B are expressed, and in cortical thick ascending limb (10.3 nmol/ L), in which AT1A is almost exclusively expressed. The Ang II-induced calcium responses were totally abolished by the AT1 receptor antagonist losartan (1 mumol/L) but not by the AT2 antagonist PD 123319 (1 mumol/L). In the absence of external Ca2+, the peak phase of the response induced by 10(-7) mol/L Ang II was reduced and shortened, suggesting that a part of the [Ca2+]i increase originated from the mobilization of the intracellular Ca2+ pool. In conclusion, these results demonstrate that in the rat kidney: (1) AT1A is the predominant AT1 receptor subtype expressed in the nephron segments, (2) glomerulus is the only structure with a relatively high AT1B mRNA content, and (3) AT1A and AT1B receptor subtypes do not differ in their efficiency for the activation of calcium second-messenger system.

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