cAMP-dependent protein kinase phosphorylates gap junction protein in lens cortex but not in lens nucleus

MP70 (a 70 kDa membrane protein) is a component of the gap junctions of the young fibre cells in the lens outer cortex. In the older fibres deeper in the mammalian lens (lens nucleus), MP70 is processed to MP38 by cleavage and removal of the carboxy terminal half. It is shown here that cortical MP70, and its derivative MP64, can be phosphorylated with cAMP-dependent protein kinase. In contrast, MP38 from the lens nucleus is not phosphorylated by the enzyme. Proteolytic processing and this lens region specific phosphorylation are relevant for the future development of functional assays for lens gap junctions.     

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.     

Functional maturation of human T lymphocytes is accompanied by changes in the G-protein pattern

The putative guanine nucleotide binding (G)-protein involved in transduction of signals from the TCR/CD3 complex has not been identified. We have used a UV-photoaffinity labeling technique to covalently attach [alpha-32P]GTP to human lymphocyte and thymocyte membrane proteins. Ten bands specifically labeled with [32P]GTP were detected by SDS-PAGE and autoradiography in T lymphocyte membranes. Among these, a 40-kDa protein was identified by immunoblotting as the alpha-subunit of the adenylate cyclase-inhibiting G-protein, Gi, and two proteins of 44 and 46 kDa were identified as the alpha-subunits of adenylate cyclase stimulating G-protein (Gs). These proteins also served as substrates for ADP-ribosylation by pertussis toxin and cholera toxin, respectively. Comparison of GTP-labeled membrane proteins from immature and more mature thymocytes and blood T lymphocytes, revealed that bands of 26, 30, 34, 40, 44 and 46 kDa were absent or weakly labeled in immature thymocytes, intermediate in mature thymocytes, and strongest in blood T cells. Similar increases were seen in ADP ribosylation of the substrates for pertussis, cholera, and botulinum C3 toxin. However, corresponding quantitative changes in Gi and Gs were not detected by immunoblotting, which suggests that the increased labeling is caused by enhanced affinity of the proteins for GTP rather than by increased amount of protein during thymic maturation. A concomitant maturation of GTP-induced cAMP production was seen in the cell populations, but no such change occurred in direct activation of adenylate cyclase by forskolin. The changes in some (but not all) GTP-binding proteins during acquisition of immunocompetence indicates their importance in T lymphocyte physiology.     

Adenylate cyclase activity and cyclic AMP production in the outer and inner zones of the adrenal cortex

It has been reported that cells isolated from the inner zone of the guinea pig adrenal cortex fail to have a steroidogenic response to ACTH. To further explore this, adenylate cyclase activity of membrane particles and cAMP production by cells prepared from the inner and outer adrenocortical zones were determined. The cAMP response to ACTH and forskolin was similar for cells from both zones. Basal adenylate cyclase activity was significantly higher in the inner zone; and while absolute responses to ACTH, GppNHp, GTP, NaF, and forskolin were greater for the inner zone, relative responses were similar for the two zones. These observations suggest that the inner zone of the guinea pig adrenal cortex may have a defect in ACTH action at a step(s) beyond cAMP formation.     

A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3',5'-cyclic monophosphate

The photoaffinity probe (32P) 8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (32P) 8-N3 cAMP-binding proteins. The 8-N, cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells. Intact cells bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/10(8) cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP/mg protein and had a cAMP-PK activity of 2,206 units/10(8) cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.     

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.     

Redox status of the eye lens

The aim of this work was to study the regional variation of some antioxidant systems in calf lens. Specific lens regions of nearly same age were obtained by a microsectioning technique, and the concentration of reduced and oxidized glutathione, protein sulfhydryl groups and iron were measured in each lens region. The concentration of reduced glutathione, the major redox buffer in lens, exponentially decreased from the cortical regions to the nucleus. In contrast, the concentration of protein sulfhydryl groups gradually increased from the cortex toward the nucleus. The protein-bound disulfides remained constant throughout the lens. Iron was concentrated in the outer cortical region. The results show that the most dynamic redox-active zone in the lens is the subcapsular cortical region where the oxidant flux meets a highly reducing environment containing a potent redox catalyst.     

Redox status of the eye lens: a regional study

The aim of this work was to study the regional variation of some antioxidant systems in calf lens. Specific lens regions of nearly same age were obtained by a microsectioning technique, and the concentration of reduced and oxidized glutathione, protein sulfhydryl groups, and iron were measured in each lens region. The concentration of reduced glutathione, the major redox buffer in lens, exponentially decreased from the cortical regions to the nucleus. In contrast, the concentration of protein sulfhydryl groups gradually increased from the cortex toward the nucleus. The protein-bound disulfides remained constant throughout the lens. Iron was concentrated in the outer cortical region. The results show that the most dynamic redox-active zone in the lens is the subcapsular cortical region where the oxidant flux meets a highly reducing environment containing a potent redox catalyst.     

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.     

Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.     

Protein phosphorylation in the larval epidermis of Locusta migratoria: Role of cyclic AMP and 20-hydroxyecdysone

Cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinase (PK) activities were quantified in the wing-pad epidermis of Locusta migratoria during the fifth larval instar. A low level of cyclic nucleotide-independent PK activity was detected in this tissue, whereas no PK-C (Ca²⁺- and phospholipid-dependent enzyme) activity was found. The main cyclic nucleotide-PK activity was cAMP-PK and large changes were observed during the intermoult. Concomitant increases in cAMP-PK activity and cAMP-dependent phosphorylation of several endogenous polypeptide substrates occurred during the last part of the intermoult cycle. The most marked ³²P-incorporation was observed in a 43 kDa polypeptide. These changes could be correlated with the peak of 20-OH-ecdysone in both hemolymph and integument.In vitro incubation of wing-pads with 20-OH-ecdysone (48 h) did not change cAMP-PK activity, but there were major changes in the pattern of endogenous phosphoproteins in particular the appearance of 43 and 45 kDa phosphoproteins. Two-dimensional analysis revealed several groups of phosphorylated spots. The results indicate a close relationship between certain cAMP-dependent phosphorylated polypeptides and the action of 20-OH-ecdysone.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.     

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.     

3H-Spiroperidol binding and dopamine-stimulated adenylate cyclase: evidence for multiple classes of receptors in primate brain regions.

Studies of displacement by agonist and antagonist drugs of ³H-spiroperidol binding in brain regions of $\text{Cebus}$ and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of ³H-spiroperidol receptors are coupled to adenylate cyclase.     

Metabolism of cyclic AMP in isolated renal tubules: effects of prostaglandins and parathyroid hormone.

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) with maximal effects of PGE1 being 6-8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2-3 fold greater in tubules exposed to PTH than to PGE1. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates. The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles

A mechanism for the activating effect of alamethicin on membrane enzymes was investigated, using a purified preparation of cardiac sarcolemmal vesicles. (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities were measured. alamethicin increased ouabain-sensitive (Na+,K+)-ATPase activity of sarcolemmal vesicles 5- to 7-fold and adenylate cyclase activity 2.5- to 4-fold. Adenylate cyclase retained its sensitivity to the beta-adrenergic agonist isoproterenol after membranes were treated with alamethicin. Alamethicin caused a 4- to 6-fold increase in the number of detectable (Na+,K+)-ATPase enzymic sites, but no increase ws noted for the number of muscarinic-cholinergic receptor-binding sites. Phosphorylation of endogenous proteins of sarcolemmal vesicles by an intrinsic cAMP-dependent protein kinase activity was stimulated 5- to 7-fold by alamethicin. The regulatory subunit of the membrane-bound cAMP-dependent protein kinase was labeled with the photoaffinity probe 8-azido-adenosine 3':5'[32P]monophosphate (8-N3-[32P]cAMP), and it migrated with an apparent molecular weight of 55,000 in sodium dodecyl sulfate polyacrylamide gels. Alamethicin stimulated autophosphorylation of the regulatory subunit by [gamma-32P]ATP 6-fold and incorporation of of 8-N3-[32P]cAMP into the subunit 2.6-fold. The results suggest that alamethicin disrupts membrane barriers of sarcolemmal vesicles, which are mostly right side out, giving substrates and activators access to enzymic sites in the interior of the vesicles, while preserving functional coupling of enzymes to their effectors.     

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁) with maximal effects of PGE₁ being 6–8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2–3 fold greater in tubules exposed to PTH than to PGE₁. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates.The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

5'guanylyl-imidodiphosphate actions on adenylate cyclase in homogenates of rat cerebral cortex plus neuronal and capillary fractions

A variety of neurohumoral agents activate adenylate cyclase in homogenates of rat frontal cortex (norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 4-Me-histamine and prostaglandins E₁, E₂ and A₂). The enzyme in homogenates of isolated cortical neurons is likewise sensitive to norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 2-Me- and 4-Me-histamine, and prostaglandin F_2α. Capillary-enriched fractions from the cortex possess an enzyme that is activated by norepinephrine, isoproterenol and dopamine. Addition of 5′-guanylyl-imidodiphosphate (Gpp(NH)p) to the cortical homogenates and neuronal fractions resulted in enhanced enzyme responses to norepinephrine, isoproterenol, dopamine, 2-Me- and 4-Me-histamine and the prostaglandins E₁ and E₂. The actions of histamine and apomorphine were not increased by the GTP analog. The sensitivity of the catecholamine-induced adenylate cyclase activation in cortical capillaries was augmented by Gpp(NH)p. Thus various cellular types within the cerebral cortex may possess different receptor characteristics with respect to stimulation of adenylate cyclase by neurohormones.     

Characterization of the bovine lens plasma membrane substrates for cAMP-dependent protein kinase

cAMP-dependent protein kinase, derived from either calf lens or bovine heart, promotes the phosphorylation of three lens plasma membrane proteins of molecular mass 28 kDa, 26 kDa and 18 kDa. Correlation of the maximal level of phosphorylation of these components with the Coomassie blue staining intensity of fractionated lens membranes suggests that the phosphorylation of the 28 kDa and 18 kDa components may be approximately stoichiometric. The protein kinase substrates could be dephosphorylated by a cardiac sarcoplasmic-reticulum-bound protein phosphatase activity. The 26 k Da component comigrated with MP26, the major lens membrane component that has been localized to the lens fiber cell junction. Treatment of phosphorylated lens membranes with chymotrypsin did not suggest that any of the three major phosphorylated components was derived from the partial proteolysis of a larger phosphoprotein. After electrophoretic separation of phosphorylated proteins, treatment with N-chlorosuccinimide confirmed that there was little similarity in the structure of the three phosphoproteins. Chymotrypsin did, however, reveal a cryptic phosphorylation site in a 22 kDa fragment that appeared to be derived from MP26. Treatment of phosphorylated membranes with reducing agents resulted in the disappearance of the 28 kDa phosphorylated component and the appearance of a new phosphorylated component of 18 kDa; neither MP26 nor the original 18 kDa component was affected by such treatment. It is not clear whether the original 18 kDa phosphoprotein, present in unreduced samples, is the same as that generated with reducing agents from the 28 kDa phosphorylated lens membrane component.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.