Regulation by calmodulin of adenylate cyclase activity in anterior pituitary.

The control of adenylate cyclase activity is exerted through separate components: receptors, guanyl nucleotide-binding protein, catalytic subunit, calmodulin (CaM), and divalent cations. We examined the roles of CaM, Mg+2, and Ca+2 in the regulation of adenylate cyclase activity in plasma membranes from anterior pituitary. Adenylate cyclase activity was reduced with 2.5 mM EGTA, 125 micrograms/ml compound 48/80, and 200 microM trifluoperazine, which are known inhibitors of CaM in a variety of tissues. Mg+2, in excess of ATP, stimulated adenylate cyclase activity. Ca+2 produced a biphasic effect on adenylate cyclase activity over the concentration range of 0.1-10 mM, exhibiting inhibition up to 2.0 mM and stimulation above that. GTP, 5'-guanylylimidodiphosphate, and F- each enhanced adenylate cyclase activity, but activity stimulated after each of these agents was reduced or returned toward control values by administration of compound 48/80 or trifluoperazine. In the absence of free Mg+2 (i.e. Mg+2 in excess of ATP concentrations), 10 mM Ca+2 produced marked stimulation of adenylate activity which was not reduced by trifluoperazine. We concluded that the plasma membranes from anterior pituitary possess a CaM-dependent adenylate cyclase and that activation of adenylate cyclase by guanyl nucleotide-binding protein requires CaM. Ca+2 may have allosteric binding sites on the catalytic subunit, and Ca+2 and Mg+2 appear to have antagonistic effects at different binding sites.

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.

Properties and characterization of low molecular weight inhibitor of calmodulin-dependent cAMP phosphodiesterase from rat liver.

We have identified two different species of inhibitors of calmodulin-dependent cAMP phosphodiesterase: 1) a low molecular weight (LMW) and 2) a high molecular weight (HMW) form. These inhibitors are extracted from rat liver. Both LMW and HMW inhibitors are heat-stable, acidic in nature and lose activity with prolonged storage and/or repeated freezing and thawing. The low molecular weight inhibitor has been purified to about 7,000-fold with 300% recovery. LMW inhibits calmodulin-dependent cAMP phosphodiesterase regardless of the source of calmodulin (e.g. fat, brain, heart, erythrocytes). LMW appears to be lipid in nature with a molecular weight of 1,500-5,000. The role of these inhibitors in diabetes and mechanism of action of insulin is presented.

Studies of insulin resistance in the streptozotocin diabetic and BB rat: activation of low Km cAMP phosphodiesterase by insulin.

The streptozotocin diabetic rat (STZ-DM) has been the best animal model for the study of insulin-deficient diabetes. A spontaneous diabetic BB Wistar Rat (SDR) has now been evaluated as a model for insulin-dependent diabetes that more closely reflects this disease in humans. The authors assessed the ability of insulin to stimulate the Vmax of a low Km cAMP phosphodiesterase (PDE) in adipose tissue of control, streptozotocin diabetic (STZ-DM) rats, and spontaneous diabetic BB rats (SDR). In addition, the authors examined the effect of streptozotocin on the nondiabetic littermates of the SDR animal, the NDR rat. Insulin stimulated Vmax of low Km cAMP PDE in control rat adipose tissue by 20% at 5 minutes. Insulin also stimulated Vmax of both SDR and NDR by 50% at 5 minutes. In contrast to control and both subgroups of the BB rat (SDR and NDR), insulin stimulated adipose tissue from STZ-DM less than 10% at 5 minutes. NDR animals rendered diabetic with streptozotocin were more responsive to insulin. The data demonstrate some similarities and differences between streptozotocin-induced diabetes and spontaneous diabetes in the BB rat. Reduced responsiveness to insulin appears to be more a part characteristic of streptozotocin diabetes than diabetes in the BB rat. The absence of significant insulin resistance in the spontaneous diabetic BB rat also is more consistent with the pathophysiological mechanisms usually seen both in other insulin-dependent diabetic rat models and insulin-dependent diabetes in man. However, both animal models of diabetes, ie, STZ-DM and BB, like man, respond to insulin therapy.

Oxygen consumption and thyroid function in the squirrel monkey (Saimiri sciureus).

Thyroidal accumulation of 125I and the biological half-life of 131I-thyroxine were determined in the squirrel monkey. Oxygen consumption and the respiratory quotient were also measured. A peak uptake of 131I by the thyroid of 45.3% of the injected isotope occurred 4 hours after administration of the radioisotope. The biological half-life of the 131I-thyroxine was 22-24 hours. Oxygen consumption was 1.02 cm3 O2/g/hour and the respiratory quotient was 0.82. It was concluded that the squirrel monkey is hypermetabolic when compared with other laboratory animals of the same size.

Hormonally sensitive cyclic AMP phosphodiesterase in liver cells. An ecto-enzyme.

Ecto-cyclic AMP phosphodiesterase activity was determined from freshly isolated and cultured liver cells. The cells were capable of hydrolyzing cyclic AMP in the medium. The ecto-phosphodiesterase represents a low Km phosphodiesterase which was activated by physiological concentrations of insulin. The product, 5'-AMP, was recovered in the medium and not with the cells. The enzyme was inhibited with aminophylline and trypsin. The ecto-phosphodiesterase activity was proportional to cell number, and total phosphodiesterase activity increased 5- to 10-fold when the cells were ruptured. About one-third of the ecto-phosphodiesterase activity from freshly isolated liver was due to phosphodiesterase in the medium. No phosphodiesterase was in the medium of cultured liver cells.

Calmodulin-dependent cyclic AMP phosphodiesterase in liver plasma membranes: stimulated by insulin.

In vivo insulin consistently stimulates the plasma membrane, high-affinity (low Km) cyclic AMP phosphodiesterase (PDE) from diabetic rat liver or adipose tissue. In vitro stimulation of membrane PDE by insulin has been reported to be inconsistent. Also, the involvement of calmodulin (CaM) in insulin stimulation of PDE has been controversial. In this report, conditions for the isolation of rat liver plasma membranes containing PDE that is sensitive to in vitro insulin stimulation and the involvement of CaM in insulin stimulation of PDE were investigated. In vitro insulin raised the Vmax of the enzyme without altering its apparent Km and was dose dependent. Insulin stimulation was lost after freezing, sonication, solubilization with detergents, or storage of the membranes at 4 degrees C for 4 h after isolation. Insulin stimulation was completely blocked by the CaM antagonist compound 48/80, EGTA, or trifluoperazine. Two isoforms of membrane-bound PDE were separated by ion-exchange chromatography following solubilization of the plasma membranes. The activities of both isoforms were stimulated by exogenous CaM. Plasma membrane PDE eluted after the application of exogenous CaM plus Ca2+. The data support the concept of a critical involvement of CaM in insulin activation of liver membrane PDE.

Insulin-stimulated calmodulin gene expression in rat H-411E cells can be selectively blocked by antisense oligonucleotides.

Reduced expression of calmodulin (CaM) and decreased activity of low Km cyclic AMP (cAMP) phosphodiesterase (PDE) are associated with uncontrolled diabetes. This condition can be readily mimicked in hepatocytes cultivated in insulin-depleted medium (Solomon, et al J. Lab. Clin. Med. in press, 1994). To investigate the relationship between CaM and low Km cAMP PDE gene expression in response to insulin, we specifically blocked expression of the three CaM genes by antisense oligonucleotides under insulin-deficient and -sufficient conditions in a rat hepatoma cell line, H-411E. We observed that both the low Km cAMP PDE activity and the steady state levels of CaM mRNA were increased in response to insulin by 50 and 100%, respectively. When antisense oligonucleotide to CaM I, II or III was added to the cultures, only CaM I antisense oligonucleotide blocked insulin stimulation of both CaM I mRNA and protein with concommittant marked inhibition of insulin's expected stimulation of low Km cAMP PDE. Furthermore, in another experiment utilizing both antisense and oligonucleotide probes specific for CaM I, II, or III together, only CaM I mRNA expression was blocked. We conclude that H-411E cells respond to insulin by appropriate increases in CaM transcripts. Furthermore, the stimulatory effect of insulin on both CaM synthesis and activation of low Km cAMP PDE could be blocked by antisense to CaM I, but not II or III genes. Therefore, in addition to the above conclusions, H-411E hepatoma cells appear to be an excellent in vitro system to explore the molecular mechanisms by which CaM and low Km cAMP PDE genes are regulated in the diabetic state.

Intramuscular energy sources in dogs during physical work.

Three groups of dogs were run under different experimental conditions characterized by varying the work load or the running time. Lipid and glycogen analyses were carried out on biopsy specimens from the biceps femoris muscle before and after exercise. In addition, arterial and venous triglycerides and free fatty acids were determined on plasma samples from one group of dogs that had been previously catheterized. Under the conditions of these experiments, results revealed: (1) plasma triglycerides did not contribute significantly to the energy supply for muscle contraction; (2) plasma free fatty acid efflux into muscle was increased during mild exercise but significantly lowered during heavy exercise; (3) exercise did not affect the phospholipid level or its composition in the muscle; and (4) muscle triglyceride levels may increase, decrease, or remain unchanged, depending upon the work load imposed by the exercise.