Angiotensin II receptor subtypes involved in the modulation of purinergic and adrenergic vasoconstrictions to periarterial electrical nerve stimulation in the canine splenic artery.

Previous experiments demonstrated that periarterial electrical nerve stimulation induced a double-peaked vasoconstriction consisting of an initial transient, predominantly P2X-purinoceptor-mediated, constriction followed by a prolonged, mainly alpha1-adrenoceptor-mediated, response in the canine splenic artery. Angiotensin II at a concentration of 0.1 nM did not affect the basal vascular tone and vasoconstrictions to exogenously administered noradrenaline (0.03-3 nmol) and adenosine 5'-triphosphate (0.01-1 micromol), but it markedly potentiated the double-peaked responses to nerve stimulation. The potentiating effect of angiotensin II was inhibited by KRH-594 (10 nM), a selective angiotensin II type 1 receptor antagonist, but was not influenced by PD123319 (0.01-0.1 microM), a selective angiotensin II type 2 receptor antagonist. The results indicate that angiotensin II potentiates sympathetic purinergic and adrenergic vasoconstrictions through the prejunctional angiotensin II type 1 receptor subtype in the canine splenic artery.

Multiple angiotensin receptor subtypes in normal and tumor astrocytes in vitro.

A role for neuropeptide receptors in glial tumorigenesis has recently been proposed. Although angiotensin receptors are known to mediate proliferative effects in many cell types, including brain astrocytes, the possible participation of these receptors in glial tumorigenesis remains unknown. In the present study, we have examined the expression of the molecularly defined angiotensin receptor subtypes AT(1a), AT(1b), and AT(2) in normal perinatal rat astrocytes and in a panel of tumor adult astrocytoma cells, using the reverse transcriptase-polymerase chain reaction (RT-PCR). Subsequently, we compared the mitogenic effect of the angiotensins A(1-8), A(2-8), A(3-8) and the heptapeptide "metabolite" A(1-7), on both normal and tumor astrocytes, measured in terms of the incorporation of tritiated thymidine. Our results indicate that AT(1a), AT(1b), and AT(2) angiotensin receptor mRNA is commonly expressed by many of these cells. Of notable exception is the astrocytoma U373 which was not found to express AT(1) or AT(2) mRNA. Chronic (24-h) incubation of cells with A(1-8) and A(1-7) lead to the induction of mitogenesis, even in the AT(1) and AT(2) mRNA negative astrocytoma cell line U373. Moreover, pharmacological analysis indicated that the observed mitogenic effects are not mediated by the AT(1) or AT(2) type receptors, but rather by a novel, specific A((1-7)) angiotensin receptor, since mitogenesis was shown to be partially blocked by the A(1-7) analogue D-Ala(7)A(1-7) and by the protease inhibitor orthophenanthroline (100 microM). Using Fura-2 spectrophotometry, we found that activation of this receptor does not alter intracellular calcium levels; however, preincubation with the protein kinase kinase inhibitor U0126 (10 microM) was found to inhibit these mitogenic effects partially. Overall, these results which demonstrate that normal and tumor astrocytes express a greater variety of angiotensin receptor subtypes than previously thought, support the idea that A(1-7) and its receptor signaling system may play an important role in shaping the astrocyte population during development. Moreover, the untimely expression of this A((1-7)) receptor may represent an important etiological component in the development of brain astrocytomas.

[Angiotensin II receptor subtype in human adrenal glands].

Although adrenal gland is one of the major target organs of angiotensin II (Ang II), the pathophysiological significance of the its receptor subtype has not been elucidated. We demonstrated by reverse transcription-polymerase chain reaction with Southern blot analysis mRNA expression of both AT1 and AT2 in human adrenal tissues of normal adrenocortical tissues, aldosterone-producing adenoma, Cushing's syndrome, and pheochromocytoma. Ang II-induced aldosterone secretion in vitro was suppressed only by 50% in the presence of selective AT1 antagonist CV-11974, while AT2 agonist CGP-42112 increased aldosterone secretion by 55% over the control. Ang II or CGP-42112 did not affect cortisol secretion. In addition, Ang II could stimulate aldosterone secretion in AT1a knockout mice both in the presence and absence of CV-11974. These results suggest that non-AT1 receptor subtype(s) including AT2, as well as AT1, is involved in the stimulation of aldosterone secretion from human adrenals.

The angiotensin II binding site on Mycoplasma hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

Angiotensin II (AngII) binding sites were characterized on rat pheochromocytoma cells (PC-12) which are known to express exclusively the type-2 (AT2) AngII receptor. Interestingly, we found that, on confluent PC-12 cells, only partial inhibition of 125I-AngII binding was achieved when cells were incubated with a saturating concentration of PD-123 319 (an AT2 selective ligand) suggesting the presence of an atypical binding site. In binding experiments, AngII exhibited high affinity for this atypical binding site with a dissociation constant (Kd) of 16 nM. Moreover, bacitracin potently inhibited PD-123 319-resistant 125I-AngII binding with an IC50 half-maximal inhibitory concentration of 44 microM. Enzyme immunoassay revealed that the cells were contaminated with Mycoplasma hyorhynis. Contaminated PC-12 cells were photolabeled with 125I-[p-benzoylPhe1]AngII and covalently labeled proteins were subjected to polyacrylamide gel electrophoresis followed by autoradiography. Under these conditions, two distinct labeled species of 140 kilodaltons (kDa) and 95 kDa were detected. Deglycosylation of the 140 kDa-labeled AT2 receptor with glycopeptidase-F (PNGase-F) resulted in a 35 kDa protein whereas the 95 kDa band was not affected by digestion with the endoglycosidase. Thus, our results show that the AngII binding site on M. hyorhynis is structurally distinct from mammalian AT1 and AT2 receptors.

Characterization of angiotensin receptors on human skin fibroblasts.

Angiotensin II is involved in blood pressure regulation, cell growth and angioneogenesis. The angiotensin receptors which mediate the intracellular effects of angiotensin II are expressed in numerous tissues and cell types. We studied the expression of angiotensin II receptors in cultured human skin fibroblasts derived from a skin biopsy. Angiotensin II binding characteristics were analyzed by radioligand binding assays. The DNA synthesis was assessed by [H]thymidine incorporation assays. Intracellular calcium concentrations were measured by fura-2 spectrofluorometry, and mRNA expression levels were analyzed by northern blot technology. Two distinct angiotensin receptors were detectable on human skin fibroblasts: the AT1 receptor with Kd = 1.0 +/- 0.7 nmol/l and Bmax = 17.9 +/- 0.9 fmol/mg protein, and an angiotensin(1-7) binding site with Kd = 26 +/- 6.6 nmol/l and Bmax = 80.4 +/- 3.5 fmol/mg protein, as shown by competition binding assays using selective angiotensin II receptor antagonists and the heptapeptide angiotensin(1-7). The angiotensin AT1 receptor mRNA was substantially expressed in human skin fibroblasts and was subjected to homologous downregulation. In human skin fibroblasts angiotensin II caused a profound increase in intracellular calcium which was blocked by angiotensin AT1 receptor antagonists such as Exp-3174. Furthermore, both angiotension II and angiotensin(1-7) led to increased DNA synthesis in human skin fibroblasts. In conclusion, cultured human skin fibroblasts express angiotensin AT1 receptors and a putatively new angiotensin receptor activated by angiotensin(1-7), both coupled to signaling pathways involved in DNA synthesis.

Expression of angiotensin II and its receptor subtypes in endometrial hyperplasia: a possible role in dysfunctional menstruation.

Dysfunctional uterine bleeding is associated with hyperplastic endometrium, and angiotensin II may affect cyclic menstruation. Cellular distribution of angiotensin II and its receptor subtypes (AT1 and AT2) in hyperplastic endometria from patients who had dysfunctional uterine bleeding with or without progestogen treatment was investigated by immunocytochemistry and quantitative receptor autoradiography. Angiotensin II-like immunoreactivity decreased in the hyperplastic endometrial stroma and glandular epithelia compared with normal cyclic endometria. The pattern of angiotensin II immunostaining on perivascular stromal cells in hyperplastic endometria was markedly different from that detected in the normal endometrium. The angiotensin II-like immunostaining was more intense in the progestogen-treated endometria compared with normal endometria. In the progestogen-treated endometrium from patients who had regular menstrual cycles, the angiotensin II-like immunostaining was localized in the perivascular stromal cell, as seen in the normal cyclic endometrium. Both AT1 and AT2 receptor levels in the hyperplastic and progesterone-treated endometria were significantly lower than the levels detected in normal endometrium. The results suggest that the normal function of angiotensin II in endometrium may be essential for regular cyclic menstruation and that alteration in the distribution of angiotensin II and/or the levels of its receptors are likely to be involved in dysfunctional uterine bleeding associated with hyperplastic endometria.

Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements.

Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate.

Localization of the angiotensin II and its receptor subtype expression in human endometrium and identification of a novel high-affinity angiotensin II binding site.

Angiotensin (ANG) II is not only a potent vasoconstrictor but may also be involved in the regeneration of new blood vessels. In proliferative endometrium, ANG II-like immunoreactivity was detected in glandular epithelium and stroma with negligible staining around the vascular endothelium. In contrast, in secretory endometrium intense immunostaining was seen in the perivascular stromal cells around the endometrial spiral arterioles with negligible staining of the other cell types. Quantitative receptor autoradiography using the nonselective radioligand [125I]-ANG II and subtype selective competing compounds showed that endometrium contained predominantly AT2 receptors, with relatively low expression of AT1 receptors and a novel non-AT1/non-AT2 angiotensin II recognition site that was insensitive to AT1 or AT2 selective ligands. Levels of specific [125I]-ANG II receptor binding displayed cyclic changes during the menstrual cycle, reaching a maximum in early secretory endometrium and then decreasing in mid to late secretory endometrium to levels seen in early to mid proliferative endometrium. In situ hybridization showed AT1 receptor mRNA expression in the glands and in the endometrial blood vessels. The cyclic changes in ANG II-like immunoreactivity together with expression of both the known and the novel AT receptor subtypes imply that this octopeptide may play a dual role both in the control of the uterine vascular bed and also in the regeneration of the endometrium after endometrial shedding, acting as an angiogenic and mitogenic mediator.

Angiotensin-II stimulates estradiol secretion from human placental explants through AT1 receptor activation.

A complete renin-angiotensin system has been shown to be present in human placenta, but its physiological role is poorly known. To investigate the implication of this system in the regulation of steroid hormone secretion, we studied the effect of angiotensin-II on the release of estradiol and progesterone from human placental explants. Our experiments showed that angiotensin-II stimulated estradiol secretion from term placental explants in a dose- and time-dependent fashion, although progesterone release was unaffected. Estradiol release induced by angiotensin-II (0.2 mumol/L) was blocked by angiotensin AT1 receptor antagonist losartan in a dose-dependent manner, suggesting the involvement of the AT1 receptor subtype in the process. On the contrary, the angiotensin AT2 receptor antagonist PD123319 (1 mumol/L) or the angiotensin AT2 receptor agonist CGP42112A (1 mumol/L) had no effect. Analysis of the amount of steroid hormones in the placental tissues incubated for 12 h showed that angiotensin-II increased estradiol production by 34% compared with the unstimulated explants, whereas the total levels of the estrogen precursor androstenedione and testosterone were decreased by 30-45% in the presence of the peptide, suggesting a stimulatory effect on the aromatization step. This hypothesis was reinforced by the absence of effect of angiotensin-II on both estradiol and testosterone concentrations in the placental explants pretreated with the aromatase inhibitor 4-hydroxyandrostenedione (25 mumol/L). Progesterone synthesis was not affected by angiotensin-II. The present study indicates that angiotensin-II induces the secretion of estradiol from human placenta through the angiotensin AT1 receptor subtype activation, and this effect seems to be linked to the stimulation of local androgen aromatization.

Transcriptional regulation of the mouse angiotensin II type 2 receptor gene.

The promoter region of the mouse angiotensin II type 2 receptor gene was cloned, and the nucleotide sequences were determined. A computer homology search for a 1.5-kb promoter region showed that there are several consensus cis DNA elements such as C/EBP, NF-IL6, and AP-1 in this region. Primer extension experiments showed that there are two transcription initiation sites 16 bp apart in the mouse type 2 receptor gene. Deletion mutants of this 1.5-kb segment were prepared and fused to a luciferase reporter gene. These type 2 receptor promoter-luciferase constructs were introduced into PC12W cells, which are from a pheochromocytoma cell line expressing the type 2 receptor, and luciferase activity was measured. It showed that a DNA segment between nucleotides -1497 and -874 suppresses the promoter activity of the type 2 receptor gene and that a DNA segment between nucleotides -47 and +56 is important for the basal promoter activity of the type 2 receptor gene. This proximal segment showed very weak promoter activity when introduced into vascular smooth muscle cells. Gel mobility shift assay with nuclear extracts from PC12W cells showed the presence of three DNA binding proteins that bound to a DNA probe between nucleotides -47 and +8. One DNA binding protein was only very weakly expressed in nuclear extracts from vascular smooth muscle cells, which do not express the type 2 receptor. Two other DNA binding proteins were not observed in nuclear extracts from vascular smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, expression and regulation of angiotensin II receptors.

Angiotensin II isoform 1 (AT1) receptor cDNAs were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. A single type of receptor seems to interact with more than one type of G-protein. AT1 consists of subtypes AT1A and AT1B, and the regulation of the receptors occurs at many stages. The isoform AT2 was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by GTP analogs, it is a seven transmembrane domain receptor. It mediates the inhibition of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A; the inhibition was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms.

Differential expression of angiotensin receptor 1A and 1B in mouse.

At least two distinct genes (AT1A and AT1B) encode type 1 angiotensin II (AT1) receptors in rodents. Receptor binding and Northern blot analysis have clearly demonstrated the presence of AT1 receptors and AT1-receptor mRNA in many tissues but fail to differentiate which type 1 receptor subtype is expressed. A reverse-transcriptase polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) assay was developed to differentiate the expressed mRNA by subtype. Expression of AT1A was clearly evident in kidney, liver, adrenal gland, ovary, brain, testes, adipose tissue, lung, and heart of adult mice. AT1B was absent from most of these tissues but was detectable in brain, testes, and adrenal gland. No significant differences in expression were evident in kidney, liver, brain, lung, or heart from 16.5- or 18.5-gestation-day fetuses, and only AT1A was evident in placenta. Expression of AT1B was confirmed in adrenal gland, brain, and testes, using a primer set that specifically amplifies only AT1B mRNA. Expression of AT1A and AT1B was also examined in As4.1 cells, a renin-expressing mouse kidney tumoral cell line. Receptor binding and competition assays using AT1- and AT2-receptor antagonists revealed that only AT1 receptors are present on the cell surface. Extremely low levels of AT1-receptor mRNA was detected by Northern blot, and RT-PCR-RFLP analysis revealed that only the AT1A subtype is expressed in this cell line. Despite the high homology between the coding sequence of the AT1A and AT1B genes, they exhibit disparate tissue-specific expression profiles.

Heterogeneous expression of angiotensin II AT1 receptors in neointima of rat carotid artery and aorta after balloon catheter injury.

We examined the expression of angiotensin II receptor subtypes and angiotensin-converting enzyme in the rat aorta and carotid artery at 1, 2, 4, 8, 15, and 30 days after balloon catheter injury or sham surgery. The AT1 receptor expression was enhanced in the neointima at 8 days in the aorta and carotid artery compared to that in intact media. Maintenance of the high expression of AT1 receptors in the neonintimal tissue at 15 and 30 days was localized to a subpopulation of neointimal cell close to the lumen of the vessel and was correlated to the distribution of smooth muscle cells immunoreactive to proliferating cell nuclear antigen. During the initial stages after injury, binding of [125I]351A to angiotensin-converting enzyme was significantly decreased in both the intima/media layers as well as adventitia in carotid artery and aorta. Binding of [125I] 351A to angiotensin-converting enzyme was significantly lower in the neointima compared to that in the intima/media of intact vessels. Our results reveal that the expression of AT1 receptors is heterogeneous in the neointima, and suggest that enhanced expression of AT1 receptors in the balloon catheter-injured carotid artery and aorta may be limited to proliferating intimal smooth muscle cells.

Angiotensin II receptor subtypes on adrenal adenoma in primary hyperaldosteronism.

Patients with an aldosterone-producing adenoma (APA) characteristically fail to show an increase in plasma aldosterone (PA) concentration with maneuvers that increase angiotensin II (Ang II), yet they retain a brisk response of PA to adrenocorticotrophic hormone. Therefore, adrenal Ang II receptor binding was characterized in a patient with APA who had a blocked PA response to Ang II infusion before adrenalectomy. The binding of [125I]Sar1,IIe5-Ang II in adrenal gland and tumor was fully displaced by excess Ang II. In the tumor, 98% of [125I]Sar1,IIe5-Ang II binding was displaced by the AT, receptor antagonist losartan, yet only 5% was displaced by the AT2 receptor antagonist PD-123,319. Autoradiography of the adrenal gland itself showed a predominance of AT1 receptors in the cortex and AT2 receptors in the medulla. The tumor showed a predominance of AT1 receptors, but there was some evidence of a limited population of AT2 receptors. The tumor and adjacent adrenal contained high concentrations of Ang II. In conclusion, a defect in Ang II-stimulated aldosterone secretion in APA occurs despite high concentrations of Ang II in the adrenal and the presence of specific, high-affinity Ang II receptor binding sites.

Enhanced expression of angiotensin II receptor subtypes and angiotensin converting enzyme in medroxyprogesterone-induced mouse mammary adenocarcinomas.

Angiotensin II receptors of the AT1 subtype were very highly expressed in medroxyprogesterone-induced ductal adenocarcinomas of the mammary gland in BALB/c mice. AT1 receptors are associated only to neoplastic epithelial cells. Lobular adenocarcinomas expressed very few AT1 receptors and expressed AT2 receptors only in areas corresponding to peritumoral connective tissue. Binding to angiotensin converting enzyme was present in all adenocarcinomas studied and was higher in ductal than in lobular adenocarcinomas. Normal mammary gland did not express either angiotensin II receptors or angiotensin converting enzyme. The present results are the first demonstration of angiotensin receptor subtypes and converting enzyme in mammary adenocarcinomas differentially expressed in tumors of ductal and lobular origin. Localization of receptor subtypes could be useful to study the differentiation of mammary cells during experimental mammary carcinogenesis in mice.

Angiotensin II stimulates cellular hypertrophy of LLC-PK1 cells through the AT1 receptor.

We have investigated possible growth-promoting effects of angiotensin II (ANG II) in the pig tubular epithelial cell line LLC-PK1. 10(-6)-10(-10) M ANG II inhibited proliferation as measured by [3H]thymidine incorporation. However, the same concentration of peptide induced tubular hypertrophy as assessed by increases in de novo protein synthesis, total protein, and RNA content. These effects were mediated through AT1 receptors. Furthermore, LLC-PK1 cells exhibited specific binding sites for ANG II and expressed mature mRNA for the vascular smooth muscle AT1 receptor. ANG II incubation of cells significantly lowered intracellular cAMP, and the hypertrophogenic action of ANG II was abolished by co-incubation with the relative stable analogue dibutyryl-cAMP, suggesting the involvement of intracellular nucleotides in the signal-transduction processes leading to hypertrophy. Our results collectively suggest that ANG II is a hypertrophogenic growth factor for LLC-PK1 cells. Considering the importance of tubular hypertrophy in the progression of renal failure in many models, strategies to block the action of ANG II on tubular cells may offer an attractive alternative way to control deterioration of renal function besides modulation of haemodynamics.

Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells.

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of angiotensin II receptor subtypes on the regulation of aldosterone secretion in the adrenal glomerulosa zone in the rat.

The role of AII receptors subtypes, AT1 and AT2, in the regulation of aldosterone secretion was studied in adrenal glomerulosa cells and membranes from rats on normal and low sodium intake, using AII receptor subtype-specific antagonists. In adrenal glomerulosa cells, more than 90% of the receptors were AT1 and there was a good correlation between the potencies of the antagonists to inhibit ligand binding, and AII-stimulated aldosterone production and inositol phosphate formation. The inhibition of basal and ACTH-stimulated cAMP by AII was also abolished by the AT1, but not the AT2, antagonist. Sodium restriction for 6 days increased both receptor subtypes in the same proportion, but only the AT1 antagonist inhibited AII-stimulated aldosterone production. The data demonstrate that AT1 receptor mediates the regulatory actions of AII in the adrenal zona glomerulosa.

Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation.

A rat angiotensin, type 1A (AT1A) receptor cDNA was cloned recently and shown to be a member of the 7-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type 1 receptor (AT1B) in the rat which exhibits high similarity with the AT1A receptor relative to amino acid sequence (95% identity), binding of angiotensin II analogs, and utilization of Ca+2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT1B mRNA whereas vascular smooth muscle and lung express primarily AT1A mRNA. Estrogen treatment suppressed AT1B but not AT1A mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative AT1 receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins.

Vasopressin elevates cytosolic calcium in small cell lung cancer cells.

The ability of vasopressin to elevate cytosolic Ca2+ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca2+ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca2+ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca2+ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca2+. The response to vasopressin was strongly blocked by [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,O-MeTyr2,Arg8]vasopressin and weakly blocked by [(beta-mercapto-beta,beta-cyclopentamethylene propionic acid)1,O-MeTyr2,Orn8]vasotocin. These data suggest that V1 vasopressin receptors are present on SCLC cells.