Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins.

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961-F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2.

BMP7 and activin are members of the transforming growth factor beta superfamily. Here we characterize endogenous activin and BMP7 signaling pathways in P19 embryonic carcinoma cells. We show that BMP7 and activin bind to the same type II receptors, ActRII and IIB, but recruit distinct type I receptors into heteromeric receptor complexes. The major BMP7 type I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRIB). BMP7 and activin elicited distinct biological responses and activated different Smad pathways. BMP7 stimulated phosphorylation of endogenous Smad1 and 5, formation of complexes with Smad4 and induced the promoter for the homeobox gene, Tlx2. In contrast, activin induced phosphorylation of Smad2, association with Smad4, and induction of the activin response element from the Xenopus Mix.2 gene. Biochemical analysis revealed that constitutively active ALK2 associated with and phosphorylated Smad1 on the COOH-terminal SSXS motif, and also regulated Smad5 and Smad8 phosphorylation. Activated ALK2 also induced the Tlx2 promoter in the absence of BMP7. Furthermore, we show that ALK1 (TSRI), an orphan receptor that is closely related to ALK2 also mediates Smad1 signaling. Thus, ALK1 and ALK2 induce Smad1-dependent pathways and ALK2 functions to mediate BMP7 but not activin signaling.

Identification of a functional Gs protein in Euglena gracilis.

We found a Gs protein coupled to adenylyl cyclase in a free-living protist, Euglena gracilis. This Gs protein of approximately 42 kDa is substrate for cholera toxin and is recognized by an antibody against the C-terminal decapeptide of Gs. Furthermore, this protein is coupled to adenylyl cyclase, as shown by: (a) the activation of the enzyme by GTP-analogues and (b) the effect of cholera toxin on cAMP accumulation in intact cells and the continuous activation of adenylyl cyclase activity in membranes. These data indicate that the Gs-adenylyl cyclase-coupled system is already apparent in the protist kingdom.

Effect of okadaic acid on hormone- and mastoparan-stimulated phosphoinositide turnover in isolated rat hepatocytes.

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angiotensin II and vasopressin. Mastoparan, a peptide toxin from wasp venom that mimics receptors by activating G-proteins, also stimulates the accumulation of inositol phosphates in hepatocytes. Interestingly, this action of mastoparan was also inhibited by okadaic acid. Our data indicate that okadaic acid inhibits the phosphoinositide turnover signal transduction system in hepatocytes at a level distal to the receptors.

Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin.

The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2+, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPTs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.

Modulation of Gs activity by phorbol myristate acetate in rat hepatocytes.

Activation of protein kinase C promotes heterologous desensitization of hepatic adenylate cyclase. The basis for this desensitization was explored by use of a strategy with several independent approaches. Although not influencing the amount of forskolin-stimulated adenylate cyclase activity (catalyst), treatment with phorbol 12-myristate 13-acetate (PMA) decreased adenylate cyclase activation in response to either sodium fluoride or guanylyl imidodiphosphate [Gpp(NH)p]. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cholera toxin-treated hepatocytes and both the basal and GTP-stimulated adenylate cyclase activity of membranes from toxin-treated cells displayed a marked reduction in response to PMA. The ability of cholate extracts of hepatocyte membranes to reconstitute beta-adrenergic-stimulated adenylate cyclase activity of membrane of S49 mouse lymphoma cyc- cells was reduced by treatment with PMA. Cholera toxin-catalyzed labeling of Gs alpha-subunits was likewise diminished by phorbol ester treatment. Immunoblots of membranes from control or PMA-treated hepatocytes showed no difference in the amount of Gs alpha. Immunoprecipitation studies failed to detect phosphorylation of this G protein alpha-subunit. The data demonstrate that PMA induces an alteration in the functional status of Gs without altering the amount of this transmembrane signaling element. The alteration in Gs function may play a significant role in heterologous desensitization.

Melittin stimulates liver glycogenolysis and the release of prostaglandin D2 and thromboxane B2.

Melittin stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect was rapid and associated with production and release of prostaglandin D2 and thromboxane B2. Indomethacin blocked the release of these eicosanoids and the stimulation of glycogenolysis induced by melittin. Ibuprofen blocked the release of prostaglandin D2 induced by melittin and markedly attenuated that of thromboxane B2. Interestingly, the initial burst of glucose output induced by melittin was not inhibited by ibuprofen, although the duration of the glycogenolytic action of the peptide was greatly diminished.

Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism.

Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.

Homologous and heterologous beta-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin.

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331-336) and this heterologous beta-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous beta-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.