Activation of a beta-adrenergic-sensitive signal transduction pathway by the secosteroid hormone 1,25-(OH)2-vitamin D3 in chick heart.

In recent studies we have established that 1 alpha, 25-dihydroxy-vitamin D3[1,25(OH)2D3] rapidly stimulates dihydropyridine-sensitive calcium channel-mediated Ca2+influx in chick cardiac muscle by a non-genomic action which is accompanied by PKA-dependent phosphorylation of a 45 kDa microsomal membrane protein. To investigate the signal transduction pathway activated by 1,25(OH)2D3 in heart, we have compared the effects of the secosteroid hormone with those of the beta-adrenergic agonist isoproterenol (IPT) by employing cultured chick embryonic cardiac cells (myocytes) and thin-slice preparations of differentiated adult heart muscle. The increases in 45Ca2+ uptake and intracellular calcium ([Ca2+]i), cyclic AMP accumulation and changes in microsomal protein phosphorylation evoked by 1,25(OH)2D3 could be reproduced by IPT. When combined treatments with the sterol and the beta-adrenergic agonist were performed, no additive stimulation of these parameters was observed, suggesting that a common signal transduction pathway mediates the effects of 1,25(OH)2D3 and IPT. The participation of a guanine nucleotide binding protein (G protein) in the 1, 25(OH)2D3-induced changes in heart was investigated. AlF4(-), an activator of G proteins, and cholera and pertussis toxins, like 1, 25(OH)2D3 increased 45Ca2+ uptake by myocytes. AlF4(-) did not further stimulate the effects of 1,25(OH)2D3 thereby showing that a G protein is involved in the hormone action. Moreover, 1,25(OH)2D3 potentiated pertussis toxin but was unable to modify choleric toxin-dependent myocyte Ca2+ influx. Altogether, these results provide evidence indicating that the non-genomic action of 1,25(OH)2D3 on cardiac muscle calcium influx involves modulation of the beta-adrenergic-sensitive adenylyl cyclase/cAMP/PKA pathway coupled to a Gs protein.

Studies suggesting the participation of protein kinase A in 1, 25(OH)2-vitamin D3-dependent protein phosphorylation in cardiac muscle.

We have previously established that the secosteroid hormone 1alpha, 25-dihydroxy-vitamin D3 [1,25(OH)2D 3] rapidly stimulates dihydropyridine-sensitive calcium channel-mediated Ca2+ influx in chick cardiac muscle by a non-genomic action which is accompanied by phosphorylation of microsomal proteins. In the present study, we investigated the participation of the cyclic AMP/protein kinase A (PKA) signalling pathway in hormone-induced changes on protein phosphorylation in chick heart tissue. A major increase in the phosphorylation of a microsomal protein of 45 kDa, and, to a lesser extent, of a protein of 70 kDa, was observed after incubation with [gamma-32P]ATP of membranes isolated from heart thin slices (HTS) pretreated for 1-5 min with 1,25(OH)2D3. This effect was dose- and time-dependent, reaching a maximum after 3 min and at the physiological concentrations of 10(-10) and 10(-11) M. 1,25(OH)2D3 steadily increased cellular cAMP levels as a function of the dose (10( -12)-10(-9) M). The specific agonist of PKA, Sp-cAMPS and the PKA catalytic subunit stimulated the phosphorylation of the same membrane proteins as the hormone. The 1alpha,25-dihydroxy-vitamin D3-dependent changes in microsomal protein phosphorylation were diminished by the specific PKA inhibitor, Rp-cAMPS. In addition, the PKA activity ratio (-cAMP/+cAMP) increased 60% above the control after treatment of HTS with 10(-11) M 1,25(OH)2D3. The data obtained clearly indicate that activation of the cAMP/PKA signalling pathway mediates the stimulation of protein phosphorylation by 1alpha, 25-dihydroxy-vitamin D3 in chick cardiac muscle.

Conversion of human interferon-beta from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus.

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3'-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-beta (hu-IFN-beta). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-beta cDNA secreted the protein to the conditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-beta cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-beta were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hy-IFN-beta. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-beta does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-beta directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.