Role for calcium in the development of ovarial patency in Heliothis virescens

Insect oocytes sequester nutritive proteins from the hemolymph under the regulation by juvenile hormone (JH), in a process called patency. Here, a pharmacological approach was used to decipher the role for calcium in ovarial patency in the moth, Heliothis virescens. Follicular epithelial cells were exposed in calcium-free or calcium-containing media to JH I, JH II or JH III alone, or in combination with various inhibitors of signal transduction. Protein kinase inhibitors, Na(+)/K(+) -ATPase inhibitor, ouabain, an inhibitor of voltage-dependent calcium channels in plasma membrane, omega-Conotoxin MVII, endoplasmic reticulum (ER) Ca(2+) -ATPase inhibitor, thapsigargin, ER inositol 1,4,5-triphosphate receptor (IP(3)R) inhibitor, 2-ABP and ER ryanodine receptor (RyR) inhibitor, ryanodine, were used. The results of our study suggest that JH II evokes patency via protein kinase C-dependent signaling pathway, and activation of Na(+)/K(+) -ATPase, similar to JH III. Response to JH II and JH III predominantly relies upon external and internal calcium stores, using voltage-dependent calcium channels, IP(3)Rs and RyRs. In contrast, regulation of patency by JH I appears to be largely calcium independent, and the calcium-dependent component of the signaling pathway likely does not use IP(3)Rs, but RyRs only. The JH II, JH III and calcium-dependent component of JH I signaling pathway probably utilize calcium/calmodulin-dependent kinase II for activation of Na(+)/K(+) -ATPase.     

Calcitonin gene-related peptide stimulates intracellular cAMP via a protein kinase C-controlled mechanism in human ocular ciliary epithelial cells.

Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C.     

Calcium and cAMP are second messengers in the adipokinetic hormone-induced lipolysis of triacylglycerols in Manduca sexta fat body

We have previously shown that stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn -1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body. After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP. Forskolin (0.1 micrometer), an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids. Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body. Our findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers.     

Angiotensin II and dopamine modulate both cAMP and inositol phosphate productions in anterior pituitary cells. Involvement in prolactin secretion

Despite their opposite effects on prolactin secretion, both dopamine and angiotensin II inhibit adenylate cyclase activity in homogenates of anterior pituitary cells in primary culture. Dopamine and angiotensin II inhibition of adenylate cyclase was not additive, suggesting that both neurohormones inhibit the adenylate cyclase of the lactotroph cells. Pretreatment with Bordetella pertussis toxin (islet activator protein) completely suppressed the dopamine-induced inhibition of both adenylate cyclase and prolactin secretion. The islet activator protein also reversed the angiotensin II-induced inhibition of the adenylate cyclase activity. In contrast, angiotensin II stimulation of prolactin release was not affected by the toxin. Angiotensin II also induced a dose-dependent stimulation of inositol phosphates (250%) with an EC50 of 0.1 nM, close to that observed for prolactin secretion. Islet activator protein pretreatment did not block the stimulation of inositol phosphate production. Dopamine inhibited the angiotensin II-stimulated prolactin release and the production of inositol phosphates induced by angiotensin II. It is concluded that angiotensin II and dopamine receptors of lactotroph cells are able to modulate both cAMP and inositol phosphate production. The dopamine receptor of lactotrophs appears to be the first example of a receptor which is negatively coupled to the production of inositol phosphates.     

Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function

A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP-dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non-hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors.     

Nitric oxide inhibits cAMP-dependent CFTR trafficking in intestinal epithelial cells

Nitric oxide is produced during intestinal inflammation and inhibits the epithelial responsiveness to cAMP-dependent secretagogues. The effect is presumably due to inhibition of activation of the CFTR. However, because insertion of CFTR into the epithelial apical membrane is also a cAMP-dependent process, we tested the hypothesis that NO could inhibit cAMP-dependent CFTR trafficking. SCBN intestinal epithelial cells were treated with forskolin to activate adenylate cyclase activity. The cells were fixed at various times and immunostained for CFTR. Some cells were pretreated with the nitric oxide donor PAPA-NONOate, the protein kinase A inhibitor H89, or the microtubule blocker nocodazole. Cross sections of epithelial monolayers were then studied under fluorescence, and the ratio of apical to basolateral CFTR immunoreactivity was determined. Stimulation of adenylate cyclase activity caused an increase in the apical-to-basolateral ratio of CFTR within 30 s. This effect was transient and preceded changes in short-circuit current in SCBN monolayers mounted in Ussing chambers. PAPA-NONOate, H89, and nocodazole all reduced forskolin-stimulated CFTR trafficking. The inhibitory effect of the NO donor was not affected by pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. PAPA-NONOate reduced forskolin-stimulated increases in intracellular cAMP. The data suggest that a portion of the inhibitory effect of nitric oxide donors on cAMP-dependent chloride secretion is through the inhibition of cAMP-dependent insertion of CFTR into the apical plasma membrane. These data provide insight into the mechanism of secretory dysfunction in inflammatory diseases of the gut where mucosal nitric oxide is elevated.     

Properties of protein kinase and adenylate cyclase-deficient variants of a macrophage-like cell line.

Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromo-adenosine 3':5' cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growtn. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2--inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis--are mediated by a cyclic AMP-dependent portein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.     

Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol.

Cytoplasmic free calcium ([Ca2+]i) and secretion of ATP were measured in quin2-loaded human platelets. In certain conditions thrombin and collagen cause secretion while [Ca2+]i remains at basal concentrations, a response attributed to activation of protein kinase by diacylglycerol formed by hydrolysis of inositol lipids. This secretion evoked by thrombin could be totally suppressed by prostaglandin I2 or forskolin, as expected from the known ability of cyclic AMP to inhibit phospholipase C. The secretory response evoked by collagen at basal [Ca2+]i and that evoked by exogenous diacylglycerol or phorbol ester, direct activators of protein kinase-C, were much less affected by these inhibitors, suggesting that thrombin and collagen may promote formation of diacylglycerol by different mechanisms.     

Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.     

Restoration of ocular dominance plasticity mediated by adenosine 3',5'-monophosphate in adult visual cortex.

Noradrenaline (NA)-stimulated beta-adrenoreceptors activate adenylate cyclase via excitatory G-proteins (Gs). Activated adenylate cyclase in turn promotes the production of cAMP. Critical roles of cAMP-dependent protein kinase A (PKA) in divergent cellular functions have been shown, including memory, learning and neural plasticity. Ocular dominance plasticity (ODP) is strongly expressed in early postnatal life and usually absent in the mature visual cortex. Here, we asked whether the activation of cAMP-dependent PKA could restore ODP to the aplastic visual cortex of adult cats. Concurrent with brief monocular deprivation, each of the following cAMP-related drugs was directly and continuously infused in the adult visual cortex: cholera toxin (a Gs-protein stimulant), forskolin (a Gs-protein-independent activator of adenylate cyclase) and dibutyryl cAMP (a cAMP analogue). We found that the ocular dominance distribution became W-shaped, the proportion of binocular cells being significantly lower than that in respective controls. We concluded that the activation of cAMP cascades rapidly restores ODP to the adult visual cortex, though moderately. The finding further extends the original hypothesis that the NA-beta-adrenoreceptors system is a neurochemical mechanism of cortical plasticity.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Elevation of Cyclic AMP-Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP-dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus , respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP-dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP-dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP-dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

Mechanisms of hormone-induced desensitization of adenylate cyclase

Luteinizing hormone (LH) interacts with its plasma membrane receptor to activate the formation of cyclic AMP via the regulatory GTP binding protein (Gs). This is followed by a desensitization of that same hormonal response which is caused by an uncoupling of the LH receptor from Gs. The coupling between Gs and the adenylate cyclase catalytic unit remains intact. Treatment of Leydig and other cell types with phorbol esters mimics hormone-induced desensitization. However, differences between hormone- and phorbol ester-induced desensitization have been found. In testis Leydig cells phorbol esters, as well as uncoupling the LH receptor from Gs, also inactivates the subunit of the inhibitory GTP binding protein (Gi). These studies suggested that activation of protein kinase may be involved in the hormone-induced desensitization of adenylate cyclase. Paradoxically, it has also been found that two inhibitors of protein kinase C, sphingosine and psychosine also inhibited LH-induced cyclic AMP production. These effects were mainly found during the initial stimulatory period with LH. It is suggested that activation of adenylate cyclase may require a protein kinase C-mediated phosphorylation step which is followed by further phosphorylation resulting in uncoupling of the receptor from Gs. No direct stimulation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), diacylglycerol and/or activation of protein kinase C by LH in Leydig cells has been demonstrated. An alternative mechanism of protein kinase C activation has been proposed for brain cells, i.e. that involving arachidonic acid activation of protein kinase C instead of diacylglycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Calcitonin stimulates plasminogen activator in porcine renal tubular cells: LLC-PK1

Plasminogen activators are highly selective proteases that activate the proenzyme plasminogen to the general protease, plasmin. We studied a porcine kidney cell line, originally isolated as a high producer of plasminogen activator, in which activities of cellular adenylate cyclase and cAMP-dependent protein kinase are increased in response to calcitonin. We found that salmon calcitonin, in the concentration range 0.03-300 nM, increased plasminogen activator production up to approximately 1,000-fold and concurrently inhibited cell multiplication; both of these effects were reversible. Human calcitonin was approximately 0.01 times as potent as salmon calcitonin, corresponding to potency differences observed in other biological systems. Plasminogen activator production was also stimulated by other agents that raise cellular cAMP levels such as cholera toxin, phosphodiesterase inhibitors, and vasopressin, but not to the same extent as by calcitonins. The rapidity and sensitivity of the plasminogen activator determination and other cellular responses may make it possible in the future to use this cell stain in a convenient bioassay for calcitonins and their analogues.     

Ca2+-Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb

In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 = 1.1 microM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2(+)-free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 microM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2(+)-free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 microM) that completely blocked the stimulatory effect of phorbol 12-myristate 13-acetate, an activator of Ca2+/phospholipid-dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 microM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.     

The effect of estradiol on the activity of cAMP-dependent protein kinase in cells of estradiol-dependent tumors of the rat mammary gland. The role of a thermally stable protein inhibitor of cAMP-dependent protein kinase

Stimulation of rat mammary tumour growth by estradiol is due to the activation of the adenylate cyclase system and cAMP-dependent protein kinases. A single administration of estradiol to ovariectomized rats causes a rise in the cAMP-dependent protein kinase activity in cell nuclei within the first 4-6 hours after injection. This effect is probably due to the translocation of enzymes into nuclei and an increase of their synthesis. The high level of the cAMP-dependent protein kinase activity in cell nuclei was observed in actively growing intact mammary tumours, in contrast to regressing ones in ovariectomized animals. This phenomenon can be accounted for by the decrease in the content of a thermostable protein inhibitor of cAMP-dependent protein kinases rather than by the high level of cAMP.     

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.