Specific protein phosphorylation during stimulation of amylase secretion by beta-agonists or dibutyryl adenosine 3',5'-monophosphate in the rat parotid gland

The present study was undertaken in order to examine the possible involvement of protein phosphorylation during beta-adrenergic stimulation in the rat parotid gland. Isolated parotid gland slices were stimulated by either isoproterenol or dibutyryl adenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of propranolol. Amylase output was measured as a parameter for the degree of stimulation of secretion. Stimulation of secretion by either isoproterenol or Bt2AMP was associated with phosphorylation of three protein bands as revealed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and autoradiography. The apparent molecular weights of the three proteins were 35,100 (protein I), 25,700 (protein II) and 20,400 (protein III). After cell fractionation by differential and gradient centrifugation, protein I was enriched in a light membrane fraction most likely corresponding to the plasma membrane as revealed by marker enzyme analysis. Proteins II and III were recovered in a denser fraction containing mainly mitochondria and rough microsomes. The effect of isoproterenol but not that of Bt2cAMP on phosphorylation of all three protein bands was completely abolished by propranolol. The different time course in the stimulation of amylase secretion by isoproterenol and Bt2cAMP respectively was reflected by corresponding differences in the time course of protein phosphorylation.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase released from rat parotid cells incubated in Ca2+-free medium containing 1 mM EGTA. Cells preincubated with 10 microM carbachol in Ca2+-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 microM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of 45Ca from cells preequilibrated with 45Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular 45Ca level. Inositol trisphosphate, but not cAMP, evoked 45Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca²⁺-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca²⁺-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of ⁴⁵Ca from cells preequilibrated with ⁴⁵Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular ⁴⁵Ca level. Inositol trisphosphate, but not cAMP, evoked ⁴⁵Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

Amylase secretion from saponin-permeabilized parotid cells evoked by cyclic AMP

Adenosine 3',5'-monophosphate (cAMP) evoked amylase release from saponin-permeabilized parotid cells of the rat. Saponin concentration was optimal at 10 micrograms/ml. Amylase release was stimulated by cAMP almost as well in Ca2+-free medium containing 1 mM EGTA as in the medium containing a physiological concentration of calcium. Although the basal and stimulated releases of amylase were markedly reduced by the further addition of 5 mM EGTA, the effect of cAMP was still detectable. The half-maximal dose of cAMP was 0.3 mM, whereas those of dibutyryl cAMP and 8-bromo-cAMP were 10-fold lower than that of cAMP. In the presence of 10 microM 3-isobutyl-1-methylxanthine, the half-maximal dose of cAMP was also decreased by 5-fold. These results suggest: 1) intracellular calcium is not essential for the exocytosis of amylase stimulated by cAMP; 2) the responsiveness of the cells to exogenous cAMP is reduced by phosphodiesterase.     

Calcium metabolism and amylase release in rat parotid acinar cells.

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).     

Effect of PT-treatment on ANP-mediated inhibition of adenylate cyclase and amylase release in rat parotid gland

Effects of pertussis toxin (PT) treatment on atrial natriuretic peptide (ANP)-mediated inhibition of adenylate cyclase and amylase release were investigated in rat parotid gland. Adenylate cyclase activity stimulated by GTPS in PT-treated membranes was much larger than that in normal membranes. ANP dose-dependently inhibited adenylate cyclase stimulated by GTPS in control rat parotid membranes, however in membranes prepared from PT-injected (in vivo) rat parotid gland, ANP did not inhibit adenylate cyclase. ANP(10^–7M) inhibited cAMP accumulation stimulated by forskolin (10^–6M) in control rat parotid acinar cells by about 34%, however, in PT-treated cells, the inhibitory effect of ANP was attenuated completely. In control cells, amylase release stimulated by isoproterenol (10^–6M) and forskolin (10^–6M) were also depressed by ANP (10^–7M) by 27 and 30%, respectively. The inhibitory response of ANP on amylase release was completely attenuated by PT-treatment. Gi was detected as a ADP-ribosylated 41-KDa protein by incubation of parotid membranes with PT and [-³²P]NAD. In rat parotid gland, these results suggested that ANP mediates adenylate cyclase/cAMP system and consequently reduces amylase release through ANP-C receptor coupled to Gi. (Mol Cell Biochem)139: 53–58, 1994)     

Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.

We investigated the hypothesis that extracellular Na+ is required for the rapid mobilization of Ca2+ by rat parotid cells after adrenergic stimulation. When Na+ salts in the media were osmotically replaced with either choline chloride (+atropine) or sucrose, efflux of 45Ca2+ from preloaded cells, caused by 10 microM-(-)-adrenaline, was unchanged. Similarly adrenaline stimulated 45Ca2+ uptake into cells under nonsteady-state conditions in the presence or absence of Na+. Monensin, a Na+ ionophore, was able to elicit a modest increase in 45Ca2+ efflux, compared with controls. Studies of net 45Ca2+ flux, performed under near-steady-state conditions, showed that adrenaline caused net 45Ca2+ accumulation, whereas monensin caused net 45Ca2+ release. The effect of monensin required the presence of Na+ in the incubation medium. Both 1 mM-LaCl3 and 0.1 mM-D-600 prevented adrenaline-stimulated 45Ca2+ uptake into cells, but had no effect on monensin-induced changes. We conclude that (1) the rapid mobilization of Ca2+ by adrenergic agonists seen in rat parotid cells does not require a Na+out greater than Na+in gradient and (2) the nature of the monensin effect is quite different from the adrenergic-agonist-induced response.     

Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin.

Propranolol inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyl-1-methylxanthine (IBMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10(-7) M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 min). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized cells than in intact cells. Other beta-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (beta 2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the cells prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized cells to exogenous cAMP was markedly increased, indicating that propranolol neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.     

Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.     

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.     

Adenosine 3',5'-monophosphate-dependent protein kinase and amylase secretion from rat parotid gland

Tolbutamide at a concentration of 10 mM inhibited cyclic AMP-dependent protein kinase in cell-free preparations of rat parotid glands as reported in rat adipose tissues. Incubation of rat parotid slices with 10 mM tolbutamide markedly interfered with the isoproterenol stimulation of amylase secretion. A carboxy derivative of tolbutamide, 1-butyl-3-$\text{p}$-carboxyphenylsulfonylurea, had minimal inhibitory effects both on protein kinase activity and on amylase secretion. These evidences strongly suggest the participation of cyclic AMP-dependent protein kinase in amylase secretion.     

Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP.

To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half-maximum doses of isoproterenol or cAMP, and markedly inhibited their maximum effects. Okadaic acid dose-dependently increased cAMP-independent phosphorylation of some proteins and enhanced cAMP-dependent phosphorylation of 21- and 26-kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini.     

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

Evidence against direct involvement of cyclic AMP-dependent protein phosphorylation in the exocytosis of amylase.

To examine whether or not the activation of cyclic AMP-dependent protein kinase is coupled to the exocytosis of amylase from rat parotid cells, the effect of protein kinase inhibitors on amylase release and protein phosphorylation was studied. A membrane-permeable inhibitor of cyclic AMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8), and peptide fragments of the heat-stable protein kinase inhibitor [PKI-(5-24)-peptide and PKI-(14-24)-amide] strongly inhibited cyclic AMP-dependent protein kinase activity in the cell homogenate. However, H-8 had no inhibitory effect on amylase release from either intact or saponin-permeabilized parotid cells stimulated by isoproterenol or cyclic AMP. Moreover, PKI-(5-24)-peptide and PKI-(14-24)-amide did not inhibit cyclic AMP-evoked amylase release from saponin-permeabilized cells, whereas cyclic AMP-dependent phosphorylations of 21 and 26 kDa proteins in intact or permeabilized cells were markedly inhibited by these inhibitors. These results suggest that cyclic AMP-dependent protein phosphorylation is not directly involved in the exocytosis of amylase regulated by cyclic AMP.     

Evidence for the involvement of cAMP-GEF (Epac) pathway in amylase release from the rat parotid gland

Amylase release from the rat parotid gland is mainly mediated in a cAMP-dependent protein kinase (PKA)-dependent manner. In the present study, amylase release mediated in cAMP-dependent and PKA-independent manners was investigated with a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF: Epac)-selective cAMP analogue, 8CPT-2Me-cAMP. The Epac was localized in the intracellular and the plasma membrane fractions. PKA activation by 8CPT-2Me-cAMP was 100-fold lower than that by cAMP. The amylase release (% of the total) from the intact parotid acinar cells was 16 and 3.6% by isoproterenol (1microM) and 8CPT-2Me-cAMP (200microM), respectively, and that from the saponin-permeabilized cells was 15 and 3% by cAMP (100microM) and 8CTP-2Me-cAMP (10microM), respectively. H-89 inhibited cAMP-induced amylase release, but did not inhibit 8CPT-2Me-cAMP-induced amylase release. These results indicated that amylase release by beta-adrenergic stimulation is mediated through both the cAMP/PKA and cAMP/Epac signal pathways.     

Cyclic AMP antagonist Rp-cAMPS inhibits amylase exocytosis from saponin-permeabilized parotid acini.

Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.     

Inhibition of adipocyte lipolysis by papaverine: papaverine can inhibit the redistribution of hormone-sensitive lipase

Papaverine, despite being a potent phosphodiesterase inhibitor, actually blocks adipocyte lipolysis. The present study was designed to clarify the mechanism of the inhibitory effect of papaverine on lipolysis. Lipolysis, stimulated by either 10 microM isoproterenol or 5 mM dibutyryl cAMP, was significantly inhibited by papaverine (100 microM and above). Papaverine, however, did not affect the isoproterenol-induced increase in the protein kinase A (A-kinase) activity ratio. In cell-free extract from non-stimulated adipocytes, cAMP-stimulated A-kinase activities were almost completely blocked by H-89, a potent inhibitor of A-kinase, but not by papaverine. Thus, the inhibitory effect of papaverine on lipolysis could be responsible for a deficit in step(s) distal to A-kinase activity. Hormone-sensitive lipase activities in the infranatant fraction of centrifuged homogenates of cells, which were maximally stimulated with isoproterenol were significantly reduced. This result indicates that hormone-sensitive lipase redistributes from cytosol to its substrate in lipolytically stimulated cells. Papaverine completely blocked the isoproterenol-induced decrease in lipase activity in the infranatant fraction. These results suggest that papaverine blocks lipolysis through its inhibitory effect on the redistribution of hormone-sensitive lipase.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.