Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Angiotensin II stimulates receptor-mediated uptake of LDL by bovine adrenal cortical cells in primary culture

Bovine adrenal cells were isolated from the subcapsular region of the gland to obtain cultures enriched in cells of the zona glomerulosa. The cells kept in primary cultures were shown to respond to angiotensin II and adrenocorticorticotropin (ACTH) by a significant increase in aldosterone production. These primary adrenal cultures were used to study the effect of angiotensin II on LDL metabolism. Addition of angiotensin II for 48 h to the culture medium resulted in a 200-300% increase in LDL metabolism, and the lowest effective concentration was 10(-8) -10(-9) M. The angiotensin II effect became evident after 12-16 h of incubation. To compare the metabolism of the 125I-labeled protein moiety to that of cholesteryl ester of LDL, the lipoprotein was labeled also with cholesteryl linoleyl ether, a nonhydrolyzable analog of cholesteryl ester. Under basal conditions and in the presence of angiotensin II or ACTH the ratio of [3H]cholesteryl linoleyl ether to 125I indicate some preferential uptake of the cholesteryl ester moiety. Stimulation of specific LDL binding at 4 degrees C and LDL metabolism at 37 degrees C by 10(-7) M angiotensin II occurred at all concentrations of LDL studied. Linearization of the kinetic data showed that angiotensin II increased the LDL receptor number significantly but not the affinity of the LDL receptor for its ligand. The present findings indicate that in analogy to ACTH, angiotensin II can influence receptor-mediated uptake of LDL by adrenal cortical cells. It remains to be shown whether the angiotensin II effect on LDL metabolism is limited to adrenal cells or will affect other cells which express the angiotensin II receptor.     

Insulin-like growth factor II binding and action in human fetal fibroblasts

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.     

The metabolic and mitogenic effects of both insulin and insulin-like growth factor are enhanced by transfection of insulin receptors into NIH3T3 fibroblasts

Insulin and insulin-like growth factor (IGF-I) have 50% sequence homology and regulate similar cellular functions. Their membrane receptors also share 84% homology in a tyrosine kinase domain essential to transmembrane signaling and may thus share common postreceptor paths. To probe action mechanisms for these related hormones, we examined the receptor and postreceptor overlap of responses stimulated by insulin and IGF-I. NIH3T3 mouse fibroblasts have few endogenous insulin receptors and are insensitive to insulin; they have IGF-I receptors and are responsive to IGF-I. Stable transfection of these cells with cDNA for the human insulin receptor yielded a cell line (3T3/HIR) expressing greater than 6 x 10(6) receptors/cell that was highly sensitive and responsive to insulin for stimulation of deoxy[14C]glucose uptake and [3H]thymidine incorporation. The cells also showed increased responses to IGF-I, although the sensitivity was less than that for insulin. The receptor specificity of such responses was examined with a monoclonal antibody MA10 that bound to insulin receptors, but elicited no responses. When 3T3/HIR cells were preincubated with MA10, subsequent insulin- or IGF-I-stimulated deoxy[14C]glucose uptake was markedly inhibited. Likewise, the presence of MA10 caused a 10-fold increase in the concentration of insulin needed to stimulate half-maximal incorporation of [3H]thymidine and also led to diminished IGF-I-stimulated responses. These results showed that the transfected human insulin receptors coupled readily with existing effector pathways in the mouse fibroblasts and mediated metabolic and mitogenic responses to both insulin and IGF-I. Such findings indicate that insulin and IGF-I regulate common cellular functions using both overlapping receptor and postreceptor signaling pathways.     

Effects of atrial natriuretic peptide AP II on the morphofunctional state of the adrenal cortex of white rats]

The effect of atrial natriuretic peptides synthetic analog AP II on adrenal zona glomerulosa and zona fasciculata physiological regeneration have been studied on male rats. The 3H-thymidine incorporation into DNA in adrenal cortical cells was evaluated in 4 and 24 h after intraperitoneal injection of 10 or 100 mcg/kg AP II. Besides, we have investigated the influence of AP II on adrenal cortical cells karyometric parameter in 4 and 24 h and aldosterone plasma concentration in 1 h after injection. 10 mcg/kg AP II increased the fraction of labelled nuclei in zona glomerulosa and decreased the aldosterone plasma level. No significant changes were seen in zona fasciculata cells proliferation. 100 mcg/kg AP II inhibited the DNA synthesis process in adrenal zona fasciculata, but had no significant influence on zona glomerulosa physiological regeneration and aldosterone plasma concentration. No nuclear morphometric parameter changes were observed in adrenal glomerulosa and fasciculata cells of AP II--treated animals.     

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca2+ concentration, [Ca2+]i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12-O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab')2 fragment (F(ab')2 anti-Fab) on the [Ca2+]i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [3H]thymidine and/or [3H]uridine uptake. LPS stimulated a 20-120 X increase in the [3H]thymidine uptake, and a 3-7 X increase in [3H]uridine uptake without inducing any change in the [Ca2+]i. TPA induced a marginal increase in [3H]thymidine and [3H]uridine uptake, without effecting any change in the [Ca2+]i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca2+]i, but had no effect on the [3H]thymidine or [3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab')2 fragment induced sizable increases in the [Ca2+]i without affecting the [3H]nucleoside uptake. However, higher concentrations of F(ab')2 anti Fab increased the [3H]thymidine uptake and [3H]uridine uptake, while also increasing the [Ca2+]i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca2+]i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [3H]uridine uptake. Similarly, TPA virtually abolished the [Ca2+]i increase induced by the F(ab')2 anti-Fab fragment, yet it stimulated a F(ab')2 anti-Fab-induced uptake of [3H]thymidine and [3H]uridine by 120 and 10 times, respectively.     

Studies on the cell-cycle dependency of the actions of insulin-like growth factor-I in Balb/c 3T3 cells

The present study was conducted to determine the cell-cycle dependency of various actions of IGF-I in Balb/c 3T3 cells. When autophosphorylation of the IGF-I receptor was determined in [32P]-labelled cells, IGF-I increased radioactivity in a 100 K-Da phosphoprotein, presumably beta-subunit of the IGF-I receptor, both in quiescent and in primed competent cells. Likewise, IGF-I stimulated uptake of [3H]deoxyglucose independent of the cell cycle. In contrast, IGF-I increased calcium entry, radioactivity in [3H]diacylglycerol, and [3H]thymidine incorporation in primed competent cells while these reactions were not induced by IGF-I in quiescent cells. The latter three reactions were attenuated when cells were pretreated with pertussis toxin. These results indicate that some, but not all, of the actions of IGF-I are dependent on the cell cycle in Balb/c 3T3 cells. They also suggest that a pertussis-toxin-sensitive G protein may be involved in the cell-cycle-dependent actions of IGF-I.     

alpha-MSH analogues and adrenal zona glomerulosa function

Recent studies on the control of adrenal zona glomerulosa function and aldosterone secretion have focussed attention on the role of MSH-like peptides. In particular, at low concentrations, alpha-MSH has a specific stimulatory effect on rat adrenal glomerulosa cells. The synthesis of alpha-MSH analogues which have potent and prolonged effects on melanocyte systems offers new methods of examining the specificity of this response. Two peptides were tested in which potential for a beta-turn configuration was stabilised. These were: [Nle4, D-Phe7]-alpha-MSH and the cyclic [Cys4, Cys10]-alpha-MSH. In contrast to their effects on melanocyte systems, only [Cys4, Cys10]-alpha-MSH stimulated glomerulosa cells, and it was equipotent with alpha-MSH, while [Nle4, D-Phe7]-alpha-MSH and shorter fragments had no effect when added alone. [Nle4, D-Phe7]-alpha-MSH, however, augmented the response of cells already maximally stimulated with alpha-MSH and in this respect its actions resembled those of gamma-MSH and related peptides. The augmentation produced by [Nle4, D-Phe7]-alpha-MSH and gamma 3-MSH was not additive when the two peptides were added together with alpha-MSH. The results suggest that the specificity of the alpha-MSH receptors in rat adrenal glomerulosa cells and the peptide structure-function relationships in this system are different from those described for melanocytes.     

Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown.

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.     

Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture

Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats. (Payet and Isler, Cell and Tissue Res. 172, 1976; Payet and Lehoux, J. steroid Biochem. 12, 1980). We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures. These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine. Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures. The medium was changed at 24 hr intervals. The hormones were added 3 days after the culture was started. The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations. Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days. When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment. The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment. Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold. When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output. However, ACTH alone stimulated the corticosterone output 10-fold. In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells. In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures.     

Effect of a cholesterol synthesis inhibitor (BM 15.766) in the presence and absence of HDL on corticosteroidogenesis of isolated zona glomerulosa and fasciculata cells

The effect of the cholesterol synthesis inhibitor BM 15.766, 4-[2-[1-(4-chlorocinamyl)piperazin-4-yl]ethyl]-benzoic acid on the corticosteroid production was studied in order to reveal the importance of endogenous cholesterol synthesis in the function of zona glomerulosa and zona fasciculata cells of rats. Attempts were made to compensate the effect of BM 15.766 through the application of high-density lipoproteins (HDL). Electron microscopy was used to trace the binding and intracellular accumulation of colloidal gold-labelled HDL (HDL-Au, a cholesterol carrier), in the presence of the cholesterol biosynthesis inhibitor. The stimulation of both types of cells with ACTH was less effective in the presence of 2 x 10(-5) M BM 15.766. The inhibitory effect of BM 15.766 was most marked on the aldosterone production of the zona glomerulosa cells, and could not be reversed by addition of a small amount of HDL-Au. Corticosterone-aldosterone conversion was inhibited by 2 x 10(-5) M BM 15.766. ACTH-stimulated, short-term HDL uptake and internalization was not affected by the cholesterol synthesis inhibitor. The results suggest that certain metabolites of de novo cholesterol biosynthesis may participate in the control of aldosterone production.     

Differential effects of glucocorticoids on insulin-like growth factor I action in cultured human fibroblasts

Glucocorticoids act synergistically with insulin-like growth factor I (IGF-I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF-I interactive effects on human fibroblast metabolism and growth. IGF-I stimulated dose-dependent increases in early metabolic events. Half-maximal effectiveness was seen at 5–8 ng/ml IGF-I, with mean maximal responses of 1.5-, 2-, and 6-fold for [³H]2-deoxyglucose uptake, [¹⁴C]glucose incorporation, and [¹⁴C]aminoisobutyric acid (AIB) uptake, respectively. A 48-hour preincubation with 10^?7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF-I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF-I-stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF-I to fibroblast monolayers was identical in ethanol control and glucocorticoid-treated cells, with 50% displacement at ～5 ng/ml IGF-I. In addition to its synergism with IGF-I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [³H]thymidine incorporation; dexamethasone had no effect on platelet-derived growth factor or fibroblast growth factor action. Two-dimensional gel electrophoresis identified two specific glucocorticoid-induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF-I receptor-mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF-I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF-I-stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley-Liss, Inc.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha-hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M)-stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.