Codominant expression of a mutation affecting the cAMP-dependent protein kinase catalytic subunit in somatic cell hybrids of LLC-PK1 cells

The LLC-PK1 mutant cell lines FIB4 and FIB6 are affected in the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) such that they possess less than 10% parental activity. However, by Western blot analysis they were shown to possess normal levels of C subunit protein. Somatic cell hybrids were derived between mutant and LLC-PK1 cells, and examined for complementation of the cAMP-PK lesion. Codominant expression of mutant and normal alleles was observed, in that somatic cell hybrids between FIB4 and LLC-PK1, and between FIB6 and LLC-PK1 cells, exhibited cAMP-PK activity 60-75% that of LLC-PK1 cells, intermediate between mutant and normal parental cell lines. The cAMP-PK of the FIB6 x LLC-PK1 and FIB4 x LLC-PK1 hybrids was examined by ion exchange chromatography. In contrast to the FIB6 and FIB4 mutants which lack an active Type I cAMP-PK, the hybrids retained levels of active Type I cAMP-PK greater than 30% that of LLC-PK1, concomitant with the retention of catalytic activity. It was concluded that the loss of Type I kinase in the FIB6 and FIB4 mutants is most likely a consequence of the lesion in the cAMP-PK C subunit. All somatic cell hybrids examined showed levels of cAMP-PK C subunit (as determined by Western blot analysis), and in vivo regulation of cAMP-PK activation (in response to hormonal or nonreceptor-mediated stimulation of adenylate cyclase), completely comparable to those of the parental LLC-PK1 cells. Hence, no aberrant regulation of either cAMP-PK subunit levels or cAMP-PK activities was evident in the somatic cells hybrids. All data were consistent with the hypothesis that FIB4 and FIB6 contain a structural mutation affecting the cAMP-PK catalytic subunit that is expressed phenotypically in the presence of the normal allele.     

cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells.

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.     

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.     

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.     

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.     

Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.     

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.     

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.     

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition.

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.     

LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters

Mutants of the pig kidney cell line, LLC-PK1, affected in cAMP metabolism, were examined for cAMP-dependent protein kinase (cAMP-PK) activity and for cAMP-mediated induction of urokinase-type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP-PK activity and concomitantly reduced uPA production (10-20% parental) in response to calcitonin, forskolin and 8-bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP-PK and the synthesis of uPA was 40-60% parental. Thus, cAMP-mediated induction of uPA showed a dependence on the absolute levels of cAMP-PK. However, uPA synthesis in response to phorbol-12-myristate-13-acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP-PK.     

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.     

Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides

Many macrophage functions such as chemotaxis, phagocytosis, enzyme secretion, and cytotoxicity are influenced by intracellular cyclic nucleotide levels, but the regulatory mechanisms involved are poorly defined. We have developed methods that allowed us to study the activation of AC in isolated guinea pig (g.p.) macrophage membranes. AC in these membrane preparations could be stimulated approximately twofold by guanine nucleotides. We could not obtain any hormonal activation of membrane-bound AC in the absence of guanine nucleotides. In the presence of GTP, however, the hormones isoproterenol and PGE1 elicited an additional threefold rise in AC activity, which subsided after approximately 15 min. As little as 10(-8) M concentrations of these two hormones induced significant elevations of AC activity. Replacement of GTP by its nonhydrolyzable analogue Gpp(NH)p resulted in a persistent hormone-independent activation of AC, and addition of hormones enhanced this level of activation. Thus, GTP-ase activity is present in macrophage membrane preparations and serves to regulate AC activation. Hormonal stimulation of AC was receptor mediated, because the effect of the beta-adrenergic agonist isoproterenol, but not PGE1, was inhibited by the beta-adrenergic blocker propranolol. In addition, the potency series of PG corresponded to that observed for stimulation of cAMP production in intact g.p. macrophages, i.e., PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. AC activation by PG in the membrane preparation was inhibited by an alpha-adrenergic agonist, thus demonstrating one means for down regulating cAMP production in g.p. macrophages. Our studies also showed that certain hormones (e.g., beta-adrenergic agonists, PG) can exert their effect on cAMP production by stimulation of membrane-bound AC, whereas other agents such as lectins or arachidonic acid require additional intracellular components to elevate cAMP levels in macrophages. The mechanism of activation of AC by hormones in g.p. macrophage membranes appears to fit the model of a ternary complex, the components of which include the hormone receptor, AC, and guanine nucleotide regulatory protein, which transmits the signal from the receptor to AC.     

Functional compartments in cyclic nucleotide action

Cyclic AMP-dependent protein kinase (cAMP-PK) is a ubiquitous enzyme that, when activated by cAMP, is capable of phosphorylating a variety of intracellular proteins. The central postulate of cAMP-mediated hormone action is that hormones regulate intracellular cAMP concentration and cAMP-PK mediates the effects of this second messenger. Although this postulate accurately describes cAMP action in certain systems, it does not adequately provide for recent observations of the accumulation of cAMP and the activation of protein kinase without the anticipated effects on protein kinase's substrates. Both biochemical and cytochemical technics provide evidence that hormonally-specific regulation of cAMP action occurs and is important. Our thesis is that hormonal regulation of metabolic events via cAMP is localized intracellular phenomenon. We propose that occupation of some cell-surface hormone receptors leads to cAMP accumulation and the activation of protein kinase in subcellular compartments, with the consequent phosphorylation of specific, rather than all, substrates of protein kinase. circumstances potentially contributing to this specificity include: (a) physical and kinetic compartmentation of hormone-receptor-adenylate cyclase complexes non-randomly within the cell membrane; and, (b) a fixed spatial relationship of hormonally activated adenylate cyclase and specific intracellular regions by the participation of cytoskeletal proteins.     

N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V2-receptor of LLC-PK1 renal epithelial cells: an effect of concanavalin A on binding and expression.

The role of N-glycosylation in the function and biosynthesis of the vasopressin V2-receptor in LLC-PK1 renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V2-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-D-galactose-specific lectin ricin, had a marked effect on the V2-receptor in LLC-PK1 cells, increasing both receptor numbers up to twofold in vivo and specific [3H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 micrograms/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V2-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V2-receptor could be shown by the lack of a Con A effect on [3H]AVP binding in membranes prepared from LLC-PK1 cells down-regulated for the V2-receptor or from cells of the LLC-PK1 V2-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V2-receptor in LLC-PK1 cells.     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic beta-cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic beta-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic beta-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca(2+)) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca(2+) oscillations in the beta-cell. Slow Ca(2+) oscillations (<1 min(-1)) produce low-frequency cAMP oscillations, and faster Ca(2+) oscillations (>3-4 min(-1)) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca(2+) oscillations. In contrast, observed antiphasic Ca(2+) and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca(2+)-dependent AC and PDE activation in coupling of Ca(2+) and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 beta-cells.     

Characterization of cAMP-dependent protein kinase from the pupal wing epidermis of Manduca sexta

Cyclic AMP-dependent protein kinase (cAMP-PK) activity in the wing epidermis of day zero pupae of Manduca sexta was characterized. The preferred exogenous substrates were histones, subfractions H1 and H2b, casein and protamine sulfate; histone H2a was only phosphorylated moderately, while free base protamine and bovine serum albumin were poor substrates for cAMP-PK. cAMP-PK activity required Mg²⁺ and was optimal in the presence of 1 mM Mg²⁺. Co²⁺ and Mn²⁺ did not substitute for Mg²⁺, and Ca²⁺ inhibited cAMP-PK activity. The effective concentration of cAMP for activation of the cAMP-PK was substantially lower than that of cGMP (EC₅₀ 1.3 × 10⁻⁸ and 1.2 × 10⁻⁶ M, respectively). The type II isozyme of cAMP-PK comprised approx. 75% of the total cytosolic wing cAMP-PK as determined by DEAE anion exchange chromatography. Photoaffinity labeling of the whole cell homogenate with 8-azido cAMP revealed the presence of only type II isozyme. The distribution of the cAMP-PK isozymes was also determined for whole cell homogenates of brain, prothoracic glands, hemolymph, trachea, nerve cord, fat body, muscle and midgut.     

Regional distribution of the enzymes and substrates mediating the action of cAMP in the mammalian lens

Localization of adenylate cyclase activity in the outer cortical regions of the bovine lens correlates with the restriction of the Gs and Gi guanine nucleotide regulatory subunits of this enzyme to these same regions of the lens. In contrast, the major membrane substrates for cAMP-dependent protein kinase (cAMP-PK) (molecular masses of 18, 26 and 28 kDa) were identified in both the inner nuclear and the outer cortical regions of the lens. However, there were differences in the relative amounts of Pi incorporated into the 18 kDa and 28 kDa components in different lens regions. The three major membrane substrates for cAMP-PK were also phosphorylated when homogenates of lens cortex were incubated with [gamma-32P]ATP plus activators of the lens adenylate cyclase. In contrast, there was no incorporation of 32P into these substrates when homogenates of lens nucleus were used. When exogenous cAMP was added to homogenates of lens nucleus or cortex, 32P was incorporated into the membrane substrates for cAMP-PK in both regions of the lens, indicating that cAMP-PK was present in both regions. Interestingly, cAMP phosphodiesterase activity was at least 10-times greater in lens cortex than in the lens nucleus. These results indicate that while the major membrane substrates for cAMP-PK could be phosphorylated in all regions of the lens, there is a restriction of those enzymes that synthesize and degrade cAMP to the outer cortical regions of this organ.