Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function.

A novel mutant of the LLC-PK1 renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V2 receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK1 cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5'-[beta, gamma-imino] triphosphate, 8-bromo adenosine-3',5'-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK1 and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V2 receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V2 receptor gene using DNA-transfer.     

Dependence of urokinase-type-plasminogen-activator induction on cyclic AMP-dependent protein kinase activation in LLC-PK1 cells.

The activation of cyclic AMP-dependent protein kinase (cAMP-PK) in vivo was studied in LLC-PK1 pig kidney cells and the mutant cell lines M18 and FIB5, which have total levels of cAMP-PK catalytic-subunit and regulatory-subunit activities comparable with those of parental cells. The extent of cAMP-PK activation (release of active catalytic subunit from the holoenzyme) was directly correlated with the cellular cyclic AMP concentration in LLC-PK1 cells. In LLC-PK1 cells, as well as in the mutants M18 and FIB5, the extent of the induction of urokinase-type plasminogen activator (uPA) by the cyclic AMP-mediated effectors calcitonin, vasopressin and forskolin was directly correlated with the levels of activated catalytic subunit. The 'receptorless' mutant M18, which is impaired in calcitonin- and vasopressin-receptor function, did not show any activation of cAMP-PK or uPA production in response to either hormone, whereas cAMP-PK and uPA responses to forskolin were about 35% higher than in parental cells. Analysis of the FIB5-cell line revealed a lesion affecting the regulation of adenylate cyclase activity, whereby basal and stimulated (both receptor- and non-receptor-mediated) adenylate cyclase levels were less than 36% of those in parental cells. The activation of cAMP-PK in response to cyclic AMP effectors was similarly reduced, and uPA induction was concomitantly lower than that in parental cells. The results demonstrate the dependence of uPA induction by cyclic AMP effectors on dissociation of the cAMP-PK holoenzyme, implying the importance of activated free cAMP-PK catalytic subunit in this process. Thus it is concluded that the mutations in the cellular cyclic AMP-generating apparatus of the M18 and FIB5 cell lines impair uPA induction by preventing cAMP-PK activation.     

Complementation between LLC-PK1 mutants affected in polypeptide hormone-receptor function

The mutant LLC-PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP-dependent protein kinase (cAMP-PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I-labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half-maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC-PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants. Somatic cell hybrids formed between each mutant and LLC-PK1 cells exhibited normal hormone binding and activation of cAMP-PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the 'receptorless' mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC-PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Isolation and genetic characterization of a renal epithelial cell mutant defective in vasopressin (V2) receptor binding and function

A novel mutant of the LLC-PK₁ renal epithelial cell line, VPR1, was isolated after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selection using a photoactivatable vasopressin analogue [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine] vasopressin. The VPR1 mutant cell line possessed less than 5% parental V₂ receptor binding for vasopressin but exhibited normal calcitonin receptor binding. In contrast to LLC-PK₁ cells (wild type), VPR1 cells exhibited no response to vasopressin in terms of in vitro adenylate cyclase activation, in vivo cAMP production, or urokinase-type plasminogen activator induction. The responses of VPR1 cells to other agents, such as calcitonin, the adenylate cyclase activator forskolin, the GTP analogue guanosine 5′-[β,γ-imino] triphosphate, 8-bromo adenosine-3′,5′-monophosphate were comparable to those of the parental cell line. Somatic cell hybrids were derived from the cell lines LLC-PK₁ and VPR1 and analyzed for the dominance/recessiveness of the VPR1 mutant phenotype. Hybrids were found to possess normal vasopressin binding activity as well as functional responses to the hormone, indicating that the mutation affecting the V₂ receptor in VPR1 cells is recessive. The VPR1 cell line may thus have application as a recipient for the expression of the V₂ receptor gene using DNA-transfer.     

Pathway of urokinase-type plasminogen activator induction in the T47D and LLC-PK1 cell lines

Induction of urokinase-type plasminogen activator (uPA) in response to either reagents activating cAMP-dependent protein kinase (cAMP-PK) or the calcium ion phospholipid-dependent kinase (C-kinase) was compared in the LLC-PK1 and T47D cell lines. The two cell lines exhibited quantitatively different responses to calcitonin, to the phosphodiesterase inhibitor isobutylmethylxanthine, and to the adenylate cyclase activator forskolin. Both showed activation of cAMP-PK in response to all these reagents, with T47D cells displaying a greater extent of activation. T47D cells, however, failed to produce uPA in response to calcitonin, forskolin, or the cAMP analog 8-bromo-cAMP, whereas LLC-PK1 cells produced high levels of uPA in response to all these agents. Both cell lines responded to phorbol esters in terms of uPA induction, though to differing extents. Phorbol myristate acetate (PMA) was shown conclusively not to activate cAMP-PK in either cell line, even at concentrations 10-fold higher than those promoting maximal uPA induction. It was concluded that phorbol ester-mediated induction of uPA does not involve cAMP or cAMP-PK activation. These results are discussed in relation to proposed models concerning the role of cAMP-PK in uPA induction.     

Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.     

A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK₁ pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced ‘permanent’ stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK₁ cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK₁ cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.     

Relationship of (8-lysine) vasopressin receptor transition to receptor functional properties in a pig kidney cell line (LLC-PK1)

In intact LLC-PK1 cells, occupancy of vasopressin receptors (Roy, C., and Ausiello, D. A. (1981) J. Biol. Chem. 256, 3415-3522) correlated with cell cAMP production. This relationship was observed as a function of hormone dose, incubation time, and changes in receptor affinity. However, the rate of cAMP production diminished with time in intact cells exposed to high hormone concentrations, even in the presence of a phosphodiesterase inhibitor. A rapid desensitization of adenylate cyclase activity was observed in minutes upon treatment of intact cells with high hormonal concentrations. Desensitization was dose- and time-dependent. Hypertonic sodium chloride, which increased hormonal binding and cell cAMP production, prevented desensitization. The acute decrease in hormone-stimulated adenylate cyclase activity correlated with increased occupancy of low affinity binding sites. EDTA-suspended cells, which have a homogeneous population of binding sites, did not demonstrate desensitization. A proposal is made as to the consequences of this phenomenon at physiological concentrations of vasopressin.     

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.     

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Summary Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin >isoproterenol>calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin>vasopressin=PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin=calcitonin. Neither isoproterenol nor PTH had an effect.These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 1,25-dihydroxyvitamin D3 on cyclic AMP responses to hormones in clonal osteogenic sarcoma cells.

The effect of 1,25-dihydroxyvitamin D3 on adenylate cyclase responsiveness was studied in the clonal osteogenic sarcoma cell line, UMR 106-06, which responds to several bone active hormones. 1,25-dihydroxyvitamin D3 treatment had no consistent effect on basal formation of cyclic AMP in intact cells, but the responses to parathyroid hormone, isoproterenol, prostaglandin E2, salmon calcitonin and the plant diterpene, forskolin, were all attenuated, by up to 90%. The effect of 1,25-dihydroxyvitamin D3 was dose-dependent, with half-maximal effectiveness at 0.1 nM, and required 48 h treatment of cells before it became apparent. The relative potencies of other vitamin D3 compounds correlated closely with their relative affinities for the 1,25-dihydroxyvitamin D3 receptor and their biological activities in other systems. 1,25-dihydroxyvitamin D3 treatment had no effect on the kinetics of labelled calcitonin binding to UMR 106-06 cells. Furthermore, the fact that such a range of hormones was affected made a receptor mediated mechanism unlikely. Nucleotide stimulatory (Ns) unit activity was assayed after 1,25-dihydroxyvitamin D3 treatment and found to be unchanged. Islet activating protein, an inhibitor of nucleotide inhibitory unit (Ni) activity, failed to modify the 1,25-dihydroxyvitamin D3 effect. Thus the effect of 1,25-dihydroxyvitamin D3 appeared to be exerted beyond hormone receptor and nucleotide regulatory components of the adenylate cyclase complex. It is concluded that 1,25-dihydroxyvitamin D3 attenuates adenylate cyclase response to hormones by a direct or indirect action on the catalytic component of adenylate cyclase.