Angiotensin II stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells

The vasoactive peptide angiotensin II stimulates phosphorylation of myosin light chain in 32P-labeled confluent cultures of vascular smooth muscle cells derived from rat mesenteric arteries. Myosin light chain was identified and its 32P-phosphorylation level quantitated following selective immunoprecipitation with an antiserum raised against purified human uterine smooth muscle myosin. Following exposure to 0.1 nM angiotensin II, phosphorylation of the light chain peaked at 4 min and then slowly decreased. The stimulation of light chain phosphorylation at 4 min is half-maximal at approximately 0.2 mM angiotensin II; the maximal response is approximately 210% of the unstimulated level. Basal myosin light chain phosphorylation was markedly reduced by incubation of cells with dibutyryl cyclic AMP or the calmodulin-inhibitor chlorpromazine. These data suggest that angiotensin II-mediated contraction in intact blood vessels involves phosphorylation of the myosin light chain, and that phosphorylation is inhibited by a cAMP-mediated process and may be calmodulin-dependent.     

Inositol uptake in rat aorta.

The purpose of this study was to investigate the mechanism of inositol uptake into rat thoracic aorta. 3H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na(+)-dependent, and a nonsaturable, Na(+)-independent component. The Na(+)-dependent component of inositol uptake had a Km of 50 microM and a Vmax of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca2(+)-free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, an activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake into both the endothelial and smooth muscle cells. These results suggest that the uptake of inositol into vascular smooth muscle is: (1) dependent upon an inward Na(+)-gradient; (2) carrier mediated, and (3) inhibited by alpha 1 adrenoceptor agonists.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

Calcium,cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca²⁺ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca²⁺ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex.

Suspensions of renal cortical tubules were incubated with 33Pi and exposed to parathyroid hormone (40 mlg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [gamma-32P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 150 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation.

We investigated whether myosin light chain phosphatase activity changes during nitric oxide-induced relaxation of contracted intact carotid media and how changes in phosphatase activity mediate this relaxation. We also investigated one mechanism for regulating this phosphatase. Myosin phosphatase activity, myosin light chain phosphorylation, guanosine 3',5'-cyclic monophosphate (cGMP) concentration, and phosphorylation of the inhibitory protein CPI-17 were all assayed in homogenates of one carotid media ring at each time point during nitric oxide-induced relaxation. The application of sodium nitroprusside to histamine-contracted media caused rapid declines in light chain phosphorylation and force. These were temporally correlated with a rapid elevation of cGMP and a large transient increase in myosin phosphatase activity. During the early response to nitroprusside, when force declined, increases in myosin phosphatase activity, concurrent with cGMP-mediated decreases in calcium and myosin light chain kinase activity, could accelerate light chain dephosphorylation. CPI-17 was dephosphorylated upon application of nitroprusside at the same time that myosin phosphatase activity increased, suggesting that the removal of inhibition by phospho-CPI-17 contributed to the increase in myosin phosphatase activity. After 20 min of nitroprusside, myosin phosphatase activity had declined to basal levels, however low force was sustained. Additional light chain phosphorylation-independent mechanisms may be involved in sustaining the relaxation.     

Cytoplasmic Ca2+ is a primary determinant for myosin phosphorylation in smooth muscle cells

Initiation of smooth muscle contraction is associated with Ca2+/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca2+ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca2+ ionophore, ionomycin, increases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca2+ concentrations below 1 microM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca2+ concentration is apparently independent of the source of free intracellular Ca2+ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca2+. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca2+ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.     

Ca2+, calmodulin and cyclic AMP-dependent modulation of actin-myosin interactions in aorta

Incubation of bovine aortic native actomyosin with cyclic AMP and bovine aortic cyclic AMP-dependent protein kinase produced a rightward shift in the relation between free Ca2+ and both superprecipitation and actomyosin ATPase activity. The relation between free Ca2+ and phosphorylation of myosin light chains was also shifted to the right. The concentration of free Ca2+ required for half-maximal activation of both ATPase activity and myosin light chain phosphorylation was approximately 1.0 microM for control actomyosin and 2.5 microM for actomyosin incubated with cyclic AMP-protein kinase. Neither basal nor maximal activities were significantly affected by incubation with cyclic AMP-protein kinase. Addition of e microM calmodulin to cyclic AMP-protein kinase-treated actomyosin relieved inhibition of both superprecipitation and myosin light chain phosphorylation. These findings suggest that cyclic AMP-protein kinase-mediated inhibition of actin-myosin interactions in vascular smooth muscle involve a shift in the Ca2+ sensitivity of the system. This shift probably involves Ca2+-calmodulin interactions and the control of phosphorylation of the myosin light chains.     

Adenosine and 5'-chloro-5'-deoxyadenosine inhibit the phosphorylation of phosphatidylinositol and myosin light chain in calf aorta smooth muscle

Smooth muscle from calf aorta is homogenized and centrifuged. The insoluble material is subjected to sucrose density gradient centrifugation. When the heaviest fraction so obtained is incubated with radioactive ATP, two components incorporate most of the acid-insoluble radioactivity. One is a phosphoprotein with a molecular weight of 21,000. It has been identified as myosin light chain by its molecular weight, isoelectric point, and precipitation by antibody to calf aorta myosin. Its phosphorylation is strongly inhibited by EGTA, in agreement with published reports that myosin light chain kinase of smooth muscle is Ca2+ dependent. The other product is of low molecular weight, is extracted into acidic chloroform-methanol, and has been identified as phosphatidylinositol 4-phosphate. Adenosine and 5'-chloro-5'-deoxyadenosine, which are vasodilators, inhibit the phosphorylation of both substrates. Phosphorylation of phosphatidylinositol is inhibited at lower concentrations of the nucleosides than is the phosphorylation of myosin light chain. The inhibitory effects of the two nucleosides are not associated with changes in the concentration of cyclic AMP. The precise function of phosphatidylinositol phosphorylation in smooth muscle is not known, but correlations between smooth muscle contraction and increased turnover of phosphatidylinositol and its mono- and diphosphates have been reported. Myosin light chain is phosphorylated under conditions which favor smooth muscle contraction. We conclude that the inhibitory effects of adenosine described here are consistent with their physiological action as vasodilators.     

Cyclic GMP-Dependent Protein Kinase and Phosphorylation of the Endogenous Substrate Proteins in the Rabbit Superior Cervical Ganglion

When the homogenate of rabbit superior cervical ganglia (SCG) was incubated in the presence of [gamma-32P]ATP and Mg2+, two specific proteins were strongly labeled. Their apparent molecular weights were 90,000 and 54,000, respectively. The phosphorylation of the latter was significantly stimulated by 10-50 nM cyclic GMP but to a lesser extent by cyclic AMP, whereas that of the former was not stimulated significantly by either of the cyclic nucleotides. The purified protein kinase inhibitor from rabbit skeletal muscle did not inhibit the phosphorylation. These results indicated that the observed phosphorylation of 54K protein was dependent on cyclic GMP but not on cyclic AMP. When intact SCG was incubated in the presence of 32Pi, phosphorylation of 90K protein was stimulated by cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP (10 microM), whereas phosphorylation of 54K protein was not significantly stimulated by any of these substances. The present demonstration of endogenous cyclic GMP-dependent protein kinase activity and its endogenous substrate proteins raises a possibility that the physiological actions of cyclic GMP in SCG are mediated by the phosphorylation of these proteins.     

Effect of parathyroid hormone and cyclic AMP on protein phosphorylation in rabbit kidney cortex

Suspensions of renal cortical tubules were incubated with ³³P_i and exposed to parathyroid hormone (40 μg/ml) or 1 mM dibutyryl cyclic AMP. In other experiments homogenates of renal cortex were assayed for protein kinase and phosphoprotein phosphatase activity using [γ-³²P]ATP with or without 5 mM cyclic AMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphorylation of proteins measured by liquid scintillation counting of gel slices. The pattern of protein phosphorylation was similar in control tissue from both tubule suspensions and homogenates. In intact tubules, parathyroid hormone stimulated the phosphorylation of four proteins with molecular weights of approx. 1500 000, 125 000, 100 000 and 50 000 by 28%, 24%, 13%, and 20%, respectively. Results with dibutyryl cyclic AMP were comparable but more variable. Stimulation of phosphorylation by cyclic AMP in homogenates was more generalized with the major effect on a 50 000 dalton protein (50% stimulation). No effect of cyclic AMP on dephosphorylation of proteins was observed. The results are interpreted as indicating that increased phosphorylation of cell proteins is part of the cyclic AMP-mediated response of the renal cortex to parathyroid hormone.     

Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues.

Three agents that activate guanylate cyclase, sodium nitroprusside, nitroglycerin and sodium axide, were examined for their effects on cyclic GMP and cyclic AMP accumulation and muscle motility with several tissues. All of these agents, except nitroglycerin with ventricle preparations, increased cyclic GMP levels and did not alter cyclic AMP in incubations of preparations of bovine tracheal smooth muscle, guinea pig tracheal chains, taenia cecum, atria and ventricle, and rat liver and cerebral cortex. Increases in cyclic GMP with these agents occurred with relaxation of smooth muscle preparations and without alteration in the contractility of atrial preparations. These observations support the hypothesis that cyclic GMP accumulation in smooth muscle may be related to relaxation rather than contraction as proposed previously. Relaxation with these agents is not associated with alterations in cyclic AMP levels. Increases in cyclic GMP levels in atrial preparations can also occur without changes in contractile force or rate of contraction.     

Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.     

Oxytocin contracts rat uterine smooth muscle in Ca2(+)-free medium without any phosphorylation of myosin light chain

Contraction of rat uterine smooth muscle related to phosphorylation state of myosin light chain under various conditions was investigated. In the Ca2(+)-containing medium, both high K+ and oxytocin induced marked contraction of the muscle accompanied by pronounced phosphorylation of myosin light chain. In the Ca2(+)-free medium, although both vanadate and oxytocin induced slight contraction, phosphorylation of myosin light chain was only evident for vanadate but not for oxytocin. It was suggested that another mechanism distinct from myosin light chain phosphorylation might be involved in Ca2(+)-independent contraction of uterine smooth muscle elicited by oxytocin.     

Calcium and cyclic GMP regulation of light-sensitive protein phosphorylation in frog photoreceptor membranes

In frog photoreceptor membranes, light induces a dephosphorylation of two small proteins and a phosphorylation of rhodopsin. The level of phosphorylation of the two small proteins is influenced by cyclic GMP. Measurement of their phosphorylation as a function of cyclic GMP concentration shows fivefold stimulation as cyclic GMP is increased from 10(-5) to 10(-3) M. This includes the concentration range over which light activation of a cyclic GMP phosphodiesterase causes cyclic GMP levels to fall in vivo. Cyclic AMP does not affect the phosphorylations. Calcium ions inhibit the phosphorylation reactions. Calcium inhibits the cyclic GMP-stimulated phosphorylation of the small proteins as its concentration is increased from 10(-6) to 10(-3) M, with maximal inhibition of 70% being observed. Rhodopsin phosphorylation is not stimulated by cyclic nucleotides, but is inhibited by calcium, with 50% inhibition being observed as the Ca++ concentration is increased from 10(-9) to 10(-3) M. A nucleotide binding site appears to regulate rhodopsin phosphorylation. Several properties of the rhodopsin phosphorylation suggest that it does not play a role in a rapid ATP-dependent regulation of the cyclic GMP pathway. Calcium inhibition of protein phosphorylation is a distinctive feature of this system, and it is suggested that Ca++ regulation of protein phosphorylation plays a role in the visual adaptation process. Furthermore, the data provide support for the idea that calcium and cyclic GMP pathways interact in regulating the light-sensitive conductance.     

Insulin control of cyclic AMP phosphodiesterase

Cyclic AMP phosphodiesterase (PDE) is an enzyme involved in cellular homeostasis of cyclic AMP. It exists as multiple isozymes in cells, but only the high affinity, membrane-bound isozyme is sensitive to hormonal modulation. Several isozymes or isoforms of the low Km PDE have been detected. Data suggest that several mechanisms exist for hormonal modulation of PDE. Activity of the low Km PDE species may be modulated by phosphorylation/dephosphorylation, phospholipid substrate concentration, insulin second messenger, cyclic GMP, guanine nucleotide binding proteins, calmodulin, or aggregation/disaggregation of monomeric forms. Modulation of PDE isoforms by different hormones may be through different regulatory components or mechanisms.