This report shows that the cyclic AMP antagonist cyclic PIP is present in all organs and tissues of the rat so far examined: brain, heart, lung, intestine, kidney, liver, spleen, skeletal muscle and fat. The synthesis of cyclic PIP is stimulated by insulin or noradrenaline (alpha-adrenergic action) in a dose-dependent fashion. Increasing cyclic PIP synthesis with increasing insulin concentrations matches the insulin receptor binding curves. Cyclic PIP levels in blood serum remain low after hormonal stimulation and no cyclic PIP can be detected in urine. As an indication of its ubiquity, cyclic PIP was even detected in yeast. Prostaglandin E (as shown by incorporation of [3H]PGE into cyclic PIP and demonstration of a constant specific activity), myo-inositol (as shown by acid hydrolysis of the dephosphorylated cyclic PIP and mass spectrometric identification of the products) and one phosphate (as shown by the ionic nature of cyclic PIP and its inactivation by phosphodiesterase plus phosphatase) are components of cyclic PIP. Chemical derivatization experiments of cyclic PIP suggest the phosphate to be bound to myo-inositol and the myo-inositol phosphate to the prostaglandin E by its C15-hydroxyl group.

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Glucose tolerance tests in rats showed that after indomethacin treatment plasma insulin levels rose five-fold higher than in untreated controls. Accordingly, the pancreatic islets of indomethacin-treated rats secreted insulin at a threefold higher rate. Glucose tolerance tests additionally showed that indomethacin treatment led to a retarded disposal of the elevated blood glucose. Both effects appear to be caused by an attenuation of the hormone responsiveness for insulin and noradrenaline (alpha-adrenoceptor action) by indomethacin. The following observations support this view: insulin and adrenaline (alpha-adrenoceptor action) lost their ability to lower cyclic adenosine monophosphate (AMP) levels in hepatocytes; the glycogen content of liver and skeletal muscle was reduced by 95% and 65%, respectively; in adipocytes the stimulation of glucose transport by insulin was reduced by 60%. These effects of indomethacin can be reversed by the addition of exogenous prostaglandin E (PGE), as elevated cyclic AMP synthesis was again sensitive to alpha-adrenergic inhibition in the liver. These results indicate a relationship between prostaglandins and insulin action. These effects of indomethacin could result from reduced synthesis of cyclic PIP (prostaglandylinositol cyclic phosphate), a proposed second messenger for insulin and alpha-adrenoceptor action, whose synthesis was decreased by indomethacin treatment and increased by the addition of exogenous PGE. Stimulation of glucose transport by cyclic PIP was unaffected by indomethacin treatment, in contrast to the stimulation by insulin. Inhibition of PGE and cyclic PIP synthesis resulted in a metabolic state comparable to insulin resistance in non-insulin-dependent diabetes mellitus.

The biosynthesis of the functional, endogenous cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP) is performed by the plasma membrane-bound enzyme cyclic PIP synthase, which combines prostaglandin E (PGE) and activated inositol phosphate (n-IP) to cyclic PIP. The Km values of the enzyme for the substrates PGE and n-IP are in the micromolar range. The plasma membrane-bound synthase is activated by fluoride, by the stable GTP analog GMP-PNP, by protamine or biguanide, by noradrenaline, and by insulin. The activation by protamine or biguanide and fluoride (10 mM) is additive, which may indicate the presence of two different types of enzyme, comparable to phospholipase Cbeta and phospholipase Cgamma. Plasma membrane-bound cyclic PIP synthase is inhibited by the protein tyrosine kinase inhibitor tyrphostin B46 with an IC50 of 1.7 microM. However, the solubilized and gel-filtrated enzyme is no longer inhibited by tyrphostin, indicating that the activity of cyclic PIP synthase is connected with the activity of a membrane-bound protein tyrosine kinase. Cyclic PIP synthase activity of freshly prepared plasma membranes is unstable. Upon freezing and rethawing of liver plasma membranes, this instability is increased about 2-fold. Protein tyrosine phosphatase inhibitors [vanadate, fluoride (50-100 mM)] stabilize the enzyme activity, but protease inhibitors do not, indicating that inactivation of the enzyme is connected with protein tyrosine dephosphorylation. Cyclic PIP synthase is present in all tissues tested, like brain, heart, intestine, kidney, liver, lung, skeletal muscle, spleen, and testis. Apart from liver, cyclic PIP synthase activity in most tissues is rather low, but it can be increased up to 5-fold when protein tyrosine phosphatase inhibitors like vanadate are present in the homogenization buffer. Preincubation of cyclic PIP synthase of liver plasma membranes with the tyrosine kinase src kinase causes a 2-fold increase of cyclic PIP synthase activity, though this is certainly not the physiological role played by src kinase in intact cells. The data indicate that cyclic PIP synthase can be activated by two separate mechanisms: by a G protein or by protein tyrosine phosphorylation.

Reduced ability or failure to stimulate cyclic adenosinemonophosphate (AMP) synthesis on a second addition of hormone 30 min after a first stimulation was taken as an indirect indication of the synthesis of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP). In diabetic rats, because of an increased possibility of restimulating cyclic AMP synthesis, the formation of cyclic PIP should be reduced. Additionally, severalfold increased basal cyclic AMP synthesis can be observed in diabetic hepatocytes in comparison with controls. Upon measuring cyclic PIP levels after hormonal stimulation in all organs of diabetic rats, it was found that stimulation of cyclic PIP synthesis by insulin decreased gradually in a time-dependent manner. Plasma membranes were prepared from diabetic Ksj db/db mice and from spontaneously hypertensive rats (SHR), and in a subsequent assay for cyclic PIP synthetase, an up to 60% decrease of enzyme activity was found. Cyclic PIP synthetase can be completely inhibited by preincubation with protein kinase A. It is most likely that this serine phosphorylation reaction by which the enzyme is inhibited also in vivo is a result of increased cyclic AMP levels. The addition of 10(-5)-10(-4) M sulfonylureas to the enzyme assay of liver plasma membrane causes full inhibition, and the addition of 10(-5)-10(-4) M biguanides, a two- to fourfold activation of the enzyme. Activation of cyclic PIP synthetase by biguanides can also be demonstrated in intact cells. It is a fast reaction and additive with respect to the activation by fluoride or guanylyl-imidodiphosphate (GMP-PNP), and it is most likely the effect with which the biguanides produce the correcting changes in metabolism. Furthermore, antihypertensive drugs like captopril, guanethidine, and dihydralazine also activate cyclic PIP synthetase. In contrast to the activation by the biguanides, this effect is not additive to the activation by fluoride. It appears that essential hypertension and type 2 diabetes are connected with or may be the result of a reduction in synthesis of the intracellular messenger cyclic PIP, whose synthesis is stimulated by hormones like insulin and noradrenaline (alpha-adrenergic action).

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