Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.     

Immunochemical properties of lipoprotein lipase. Development of an immunoassay applicable to several mammalian species

Reuber hepatoma H-35 was found to retain the activity of carbamoyl-phosphate synthetase I. The content of this enzyme in H-35 grown in Eagle's minimal essential medium was about half that in rat liver. The enzyme from H-35 was the same as that from rat liver in molecular weight estimated by SDS-polyacrylamide gel electrophoresis, specific enzyme activity, kinetic parameters for ATP and N-acetyl-L-glutamate, and immunological crossreactivity. The enzyme in H-35 was induced by dexamethasone (1.4-fold) but not by glucagon or dibutyryl cAMP. Incorporation of [35S] methionine into the enzyme indicated that the effect of dexamethasone was due to increased synthesis of the enzyme protein (2.1-fold). By labeling with [35S]methionine, the precursor and the mature forms of carbamoyl-phosphate synthetase I were observed in the post-mitochondrial and mitochondrial fractions, respectively. By chasing the labeled cells with unlabeled methionine and cycloheximide, it was observed that the rate of translocation of the precursor into mitochondria is not affected by dexamethasone.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

The glucagon-insulin antagonism and glucagon-dexamethasone synergism in the induction of phosphoenolpyruvate carboxykinase in cultured rat hepatocytes

In hepatocytes precultured for 24 h with dexamethasone glucagon increased phosphoenolpyruvate carboxykinase activity 3-4-fold with a half maximal activity increase at 30 pM. The half maximal effective glucagon concentration was enhanced 10-fold to 300 pM when insulin was added simultaneously. The glucagon-insulin antagonism was maximally expressed when glucagon was present at low physiological concentrations. At equimolar doses it was only in the concentration range around 0.1 nM that glucagon and insulin became powerful antagonists; at higher levels glucagon was the dominant hormone. In hepatocytes not pretreated with dexamethasone glucagon still enhanced phosphoenolpyruvate carboxykinase activity, but the half maximal effective dose raised more than 30-fold to 1 nM. The degree of stimulation, however, remained essentially unchanged. Thus dexamethasone shifted the glucagon sensitivity of the cells into the physiological concentration range; it exerted a half maximal effect at 10 nM. Dexamethasone was not required for the enzyme induction proper if the cells had been pretreated with the glucocorticoid. The amount of the glucagon-stimulated enzyme induction was dependent on the time period of cell pretreatment with dexamethasone. Glucagon enhanced enzyme activity to the same constant suboptimal level irrespective of whether cells had been pretreated with glucocorticoid for 1 or for 14 h. If cells were pretreated for more than 15 h, glucagon linearly increased enzyme activity further until the maximal value was reached after 24 h pretreatment. The glucagon-insulin antagonism and the glucagon-glucocorticoid synergism were observed at physiological hormone concentrations indicating that the interaction should be effective also in vivo. Dexamethasone does not seem to be generally permissive for the inducing action of glucagon, but rather sensitizes the cell towards lower physiological hormone concentrations.     

Insulin-like growth factor I enhances proenkephalin synthesis and dopamine beta-hydroxylase activity in adrenal chromaffin cells

Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin-derived enkephalin-containing peptides and the activity of dopamine beta-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine beta-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was approximately 1 nM, with maximal effects observed at 10-30 nM. In contrast, insulin was 1000-fold less potent. Pretreatment of chromaffin cells with IGF-I increased the rate of [35S]proenkephalin synthesis 4-fold compared to untreated cells. Total protein synthesis increased only 1.5-fold under these conditions. These results suggest that IGF-I may be a normal regulator of chromaffin cell function.     

Mitogenesis in normal human fibroblasts by polyinosinic . polycytidylic acid and other synthetic acidic polymers: enhancement of action by glucocorticoids

The ability of specific synthetic polyelectrolytes to act as mitogens for quiescent normal human fibroblasts in cultures is described. Of several acidic polymers tested, polyinosinic acid .polycytidylic acid (poly I.poly C) and dextran sulfate were the most effective in stimulating 3H]thymidine incorporation (2-to 10-fold). The concentration for a half-maximal effect (ED50) was 0.4 microgram/ml (0.8 nM) for poly I.poly C, and 1.7 microgram/ml (3.4 nM) for dextran sulfate. Single-stranded polyinosinic acid or polycytidylic acid had no effect. The time course of stimulation of DNA synthesis by these acidic polymers was similar to that for naturally occurring mitogens such as epidermal growth factor, beginning at about 18 hours and reaching a maximum rate 26 to 30 hours after the addition of polymer. Glucocorticoids that have an 11-beta hydroxyl group (e.g., dexamethasone) had no effect on DNA synthesis alone, but enhanced several-fold the mitogenic activity of poly I.poly C or dextran sulfate; the ED50 for dexamethasone was 0.75 ng/ml (1.9 nM). Glucocorticoids with an 11-keto group were inactive in this respect. The labeling index following treatment of cultures with poly I.poly C and dexamethasone was 14%, compared with a labeling index of 25% following stimulation by fetal calf serum. The extent of stimulation of DNA synthesis by poly I.poly C and dexamethasone was comparable to that induced by epidermal growth factor. It appears that both the poly I.poly C and dexamethasone are required for only a short period of time (approximately 3 hours) in order to produce maximal stimulation of DNA synthesis 30 hours later.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes.

The activity changes of the urea-cycle enzymes were monitored in cultured foetal hepatocytes after dexamethasone and insulin treatments. Addition of dexamethasone induced the development of carbamoyl-phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase activities as soon as day 16.5 of gestation. When insulin was added together with dexamethasone, it markedly inhibited the steroid-induced increase in carbamoyl-phosphate synthetase, argininosuccinate synthetase and argininosuccinase activities.     

Glucocorticoid-induced insulin resistance in vitro: inhibition of insulin-stimulated methylaminoisobutyric acid uptake

We have previously developed an in vitro model for the induction of insulin resistance by glucocorticoids using 3T3-L1 fat cells (Grunfeld, Baird, Van Obberghen and Kahn 1981). In this model, glucocorticoid treatment was shown to decrease insulin binding and inhibit the acute stimulation of deoxyglucose uptake by insulin. We now extend the findings in this model to examine insulin stimulated methylaminoisobutyric acid (MAIB) uptake, an event whose expression requires m-RNA and protein synthesis and takes many hours. As previously seen with insulin stimulation of deoxyglucose uptake, one day of exposure to dexamethasone had little effect on insulin stimulation of MAIB uptake. Significant inhibition of insulin-stimulated MAIB uptake was seen after 2 days of exposure, and 3 days were required for the maximum effect of the glucocorticoid. The half-maximal concentration of dexamethasone required for inhibition was 1.6 nM. Exposure to dexamethasone produced a 57% decrease in the maximal response to insulin and a small but consistant shift in the sensitivity to insulin. As seen with the acute effects of insulin, the major locus of glucocorticoid action in inhibiting insulin stimulated MAIB uptake is also after the binding of insulin to its receptor. These data indicate that the inhibitory effects of glucocorticoids on insulin action in fat cells extend to those effects of insulin which require gene expression and are not merely limited to short-term metabolic actions of insulin.     

The specific protein phosphatase inhibitor okadaic acid differentially modulates insulin action.

The pleiotropic nature of insulin action suggests diverse mechanisms of signal transduction for the hormone. The specific protein phosphatase inhibitor, okadaic acid, is utilized to differentiate metabolic pathways that may be regulated by phosphorylation or dephosphorylation of key enzymes. In H-35 hepatoma cells, okadaic acid inhibits insulin-stimulated glycogen synthesis with an IC50 of 400 nM. In contrast, activation of lipogenesis by insulin is inhibited with an IC50 of 50 nM okadaic acid. The toxin also inhibits stimulation of lipogenesis in these cells by the insulin-sensitive inositol glycan enzyme modulator. In isolated rat adipocytes, insulin-stimulated lipogenesis is also inhibited by okadaic acid with an IC50 of approximately 1,700 nM. The antilipolytic effect of insulin in these cells is more sensitive to okadaic acid, exhibiting an IC50 of 150 nM. Maximal activation of lipogenesis by insulin is dramatically reduced by okadaic acid with no effect on the concentration required for half-maximal activation, whereas the sensitivity of insulin-induced antilipolysis is attenuated by okadaic acid, with no apparent reduction in the maximal effect of the hormone. Taken together, these data suggest that specific phosphatases may be differentially involved in some of the metabolic pathways regulated by insulin.     

Presence of Corticotropin-Releasing Factor-Stimulated Adenylate Cyclase Activity in Rat Retina

Corticotropin-releasing factor (CRF) stimulates rat retinal adenylate cyclase activity in a concentration-dependent manner. The half-maximal effect is obtained at 50 nM CRF and the maximal stimulation corresponds to approximately 90% increase of basal enzyme activity. The CRF effect is counteracted by the CRF antagonist alpha-helical CRF 9-41 with a Ki value of 40 nM. Other CRF-like peptides such as sauvagine and urotensin I are as effective as CRF with a rank order of potency of urotensin I greater than or equal to sauvagine greater than CRF. The sauvagine and urotensin I effects are not additive with that elicited by CRF. Moreover, the CRF stimulation is not additive with the increase of enzyme activity produced by vasoactive intestinal peptide or dopamine. The CRF effect is independent of the concentration of free Ca2+, is optimal at 5-10 mM MgCl2, and requires GTP. The results indicate that rat retinal adenylate cyclase is modulated by CRF via a receptor-mediated mechanism.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

The regulation of carbohydrate metabolism involves changes in the phosphorylation state of enzymes. We used okadaic acid, a potent inhibitor of protein phosphatases type 2A (IC50 0.05-2 nM) and type 1 (IC50 10-20 nM) to determine the role of these phosphatases in the control of carbohydrate metabolism by insulin in rat hepatocytes. In the absence of insulin, okadaic acid caused total inhibition of glycogen synthesis at 100 nM and half-maximal inhibition at 8-9 nM. In the presence of insulin, lower concentrations of okadaic acid (to which type 2A phosphatases are sensitive) were effective at inhibiting glycogen synthesis. 2.5 nM okadaic acid caused total inhibition of the 2-fold stimulation of glycogen synthesis by insulin but had no effect on the basal unstimulated rate of glycogen synthesis. This suggests the involvement of type 2A protein phosphatases in the stimulation of glycogen synthesis by insulin. Okadaic acid (5 nM), partially suppressed but did not abolish the increase in glucokinase mRNA levels caused by insulin, indicating that dephosphorylation mechanisms may be involved in the control of glucokinase mRNA levels by insulin. It is concluded that activation of protein phosphatases type 1 and/or type 2A by insulin may have a widespread role in the control of glucose metabolism at various sites.     

Monovalent carboxylic ionophores inhibit transport of carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells and cause accumulation of enzyme precursor

Transport of the precursor for carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells was inhibited by adding monensin or nigericin to the culture medium at a concentration of 0.5 microM, and the enzyme precursor accumulated, mainly in the cytosolic fraction. Accumulated precursor was degraded slowly with a half-life of more than 16 min. Valinomycin, nonactin, A23187, X-537A (lasalocid), bromo-lasalocid, and carbonyl cyanide m-chlorophenylhydrazone did not exhibit these effects at concentrations at which they did not inhibit protein synthesis of the cells.     

Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon. 1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK. 2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked immunoprecipitation of [35S]methionine-labelled PEPCK. 3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin. The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzyme-synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1-1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range, with glucagon being the dominant hormone.     

Acute stimulation by glucocorticoids of gluconeogenesis from lactate/pyruvate in isolated hepatocytes from normal and adrenalectomized rats

Dexamethasone stimulated gluconeogenesis from lactate/pyruvate in suspensions of hepatocytes isolated from both adrenalectomized and normal fasted rats. This stimulation was observed in incubations with 1 mM pyruvate and at a lactate/pyruvate ratio of 25 but not at a ratio of 10-13. At a lactate/pyruvate ratio of 10-13, the stimulation by dexamethasone was progressively enhanced as the pyruvate concentration was decreased to 0.25 mM. Concurrent administration of a maximally stimulating concentration of dexamethasone with angiotensin II or glucagon yielded an additive stimulation at all concentrations of the peptide hormones tested. No potentiating or permissive actions of acute glucocorticoid administration were observed using hepatocytes from either normal or adrenalectomized animals. The acute stimulation by dexamethasone was antagonized by prior addition of progesterone or cortexolone to the hepatocyte suspensions. Triamcinolone and corticosterone also stimulated gluconeogenesis. Concentrations of the active glucocorticoids needed to elicit half-maximal stimulations (Kact) were approximately 100 nM for dexamethasone and triamcinolone and 400 nM for corticosterone. Deoxycorticosterone, 17 alpha-methyltestosterone, and 5 beta-dihydrocortisol did not stimulate. Stimulation of gluconeogenesis by dexamethasone was seen following a lag averaging 9 min after the time of steroid addition. Preliminary evidence suggests that this effect was not dependent upon a stimulation of protein synthesis, but the observed stimulation and inhibition of control rates of gluconeogenesis by cycloheximide and cordycepin, respectively, demonstrate the difficulties of working with such inhibitors in attempting to answer this question.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA.

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.     

Biological effects of sulphated insulin in adipocytes and hepatocytes

Summary The binding affinity of sulphated insulin compared with unmodified, neutral insulin has been reported to be approximately four times lower in human and rat adipocytes but over twenty times lower in rat hepatocytes. In the present study the biological action of sulphated insulin was assesed in rat hepatocytes and human and rat adipocytes. To achieve half-maximal stimulation of fatty acid synthesis in rat hepatocytes about twenty one times higher concentrations of sulphated than neutral insulin were required (15.07±5.50 vs 0.71±0.34 nmol/l), this ratio being similar to the ratio of binding affinity in rat hepatocytes. In human adipocytes, half-maximal stimulation of initial rates of glucose uptake was observed at 11.6±5.1 vs 2.9±1.3 pmol/l for sulphated and neutral insulin respectively, and half-maximal inhibition of lipolysis at 31.0±13.5 vs 7.3+2.5 pmol/I respectively. These data are consistent with the four-fold lower binding affinity of sulphated insulin to human adipocytes. However, in rat adipocytes the biological potency of sulphated insulin was found to be much lower than anticipated from the binding data, half-maximal stimulation of initial rates of glucose uptake being observed at 757±299 vs 35±13 pmol/l respectively and half-maximal inhibition of lipolysis at 35.9±12.1 vs 1.5±0.5 pmol/l respectively. Thus, in rat adipocytes, approximately 22 times the concentration of sulphated insulin was required to achieve equivalent biological effect. A discrepancy between binding affinity and biological action with respect to sulphated insulin was identified in rat adipocytes but not human adipocytes nor rat hepatocytes suggesting differences in the binding-action linkage in these cells.