Beta-adrenergic regulation of a myocardial actin gene via a cyclic AMP-independent pathway.

The skeletal alpha-actin gene encodes a major component of the embryonic cardiac sarcomere that is strongly and selectively re-induced during beta-adrenoceptor-mediated hypertrophy in neonatal rat cardiac myocytes. We present evidence that beta-adrenergic induction of this gene is mediated, not by cAMP, but by a calcium-dependent pathway involving ryanodine-sensitive calcium stores. Nifedipine-induced blockade of the plasma membrane L-type calcium entry channel prevented induction of skeletal alpha-actin mRNA by isoproterenol. Activation of calcium entry by the dihydropyridine agonist Bay K8644 independently induced skeletal alpha-actin mRNA, as did cholera toxin-mediated activation of Gs. Induction of skeletal alpha-actin mRNA by compounds that directly elevate cAMP was weak relative to their effects on other cAMP-dependent phenomena and required calcium entry. In addition, selective inhibition of protein kinase A with KT5720 did not block beta-adrenergic induction of skeletal alpha-actin. Calcium ionophore A23187 did not induce skeletal actin, but prevented its induction by isoproterenol. Ryanodine had bimodal effects: 10(-10) M ryanodine induced skeletal alpha-actin mRNA, whereas 10(-6) M ryanodine prevented skeletal actin induction by beta-adrenergic stimuli. We postulate that beta-adrenergic stimulation of skeletal alpha-actin mRNA requires G-protein-coupled calcium channel activation and compartmentalized calcium release in a manner independent of the cAMP/protein kinase A signal pathway.     

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.     

A large contribution of a cyclic AMP-independent pathway to turtle olfactory transduction

Although multiple pathways are involved in the olfactory transduction mechanism, cAMP-dependent pathway has been considered to contribute mainly to the transduction. We examined the degree of contribution of cAMP-independent pathway to the turtle olfactory response by recording inward currents from isolated cells, nerve impulses from cilia and olfactory bulbar responses. The results obtained by the three recordings were essentially consistent with each other, but detail studies were carried out by recording the bulbar response to obtain quantitative data. Application of an odorant cocktail to the isolated olfactory neuron after injection of 1 mM cAMP from the patch pipette elicited a large inward current. Mean amplitude of inward currents evoked by the cocktail with 1 mM cAMP in the patch pipette was similar to that without cAMP in the pipette. Application of the cocktail after the response to 50 microM forskolin was adapted also induced a large inward current. Application of the odorant cocktail to the olfactory epithelium, after the response to 50 microM forskolin was adapted, brought about an appreciable increase in the impulse frequency. The bulbar response to forskolin alone reached a saturation level around 10 microM. After the response to 50 microM forskolin was adapted, 11 species of odorants were applied to the olfactory epithelium. The magnitudes of responses to the odorants after forskolin were 45-80% of those of the control responses. There was no essential difference in the degree of the suppression by forskolin between cAMP- and IP3- producing odorants classified in the rat, suggesting that certain part of the forskolin-suppressive component was brought about by nonspecific action of forskolin. Application of a membrane permeant cAMP analogue, cpt-cAMP elicited a large response, and 0.1 mM citralva after 3 mM cpt- cAMP elicited 51% of the control response which was close to the response to citralva after 50 microM forskolin. A membrane permeant cGMP analogue, db-cGMP elicited a small response and the response to 0.1 mM citralva was unaffected by db-cGMP. It was concluded that cAMP- independent (probably IP3-independent) pathway greatly contributes to the turtle olfactory transduction.     

Somatostatin modulation of neurally mediated pepsinogen secretion from frog esophageal mucosa

Frog esophageal mucosa contains peptic glands which are innervated by cholinergic neurons. When incubated in a medium containing 1.5 mM CaCl₂, pepsinogen release from esophageal mucosa was increased by a high potassium concentration (55 mM KCl), 1,1-dimethyl-4-phenylpiperazinium (DMPP) or bethanechol. Whereas the response to bethanechol remained little changed, the response to high KCl concentrations or DMPP was abolished in the absence of Ca²⁺. The stimulatory effects of high KCl concentrations and DMPP were also eliminated by the presence of atropine or somatostatin. Furthermore, pepsinogen release in response to bethanechol was dose-dependently inhibited by somatostatin. Frog esophagus was found to contain somatostatin-like immunoreactivity, with a higher density at the end adjacent to the stomach. Chromatography of mucosa extract on Sephadex G-50 revealed a single peak of somatostatin-like immunoreactivity that coeluted with somatostatin-14. Immunohistochemical staining of the mucosa with peroxidase antiperoxidase technique demonstrated the presence of two varieties of somatostatin-like immunoreactivity-containing cells, one individually dispersed within the intercalated septa and the other in groups within the interlobular septa of the peptic glands. These results seem to indicate that somatostatin or somatostatin-like immunoreactivity may play a modulatory role in neurally mediated pepsinogen secretion in the frog esophagus.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process.

The interaction between interleukin IL-1 alpha and PGE2 on P388D1 cells has been investigated. Preincubation of murine macrophage-like cells, P388D1, with IL-1 alpha (0-73 pM) reduced the binding of PGE2 to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1 alpha decreased the PGE2 binding by lowering both the high and low affinity receptor binding capacities (from 0.31 +/- 0.02 to 0.12 +/- 0.01 fmol/10(6) cells for the high affinity receptor binding sites and from 2.41 +/- 0.12 to 1.51 +/- 0.21 fmol/10(6) cells for the low affinity receptor binding sites). However, the dissociation constants of the receptors of the IL-1 alpha-treated cells remained unchanged. Inhibition of PGE2 binding by IL-1 alpha did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1 alpha inhibits the binding of PGE2 to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE2.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

Somatostatin inhibits prolactin secretion in the estradiol primed male rat

Somatostatin inhibits not only growth hormone secretion, but also the secretion of several other hormones. The role of somatostatin in prolactin (PRL) secretion has not been clearly demonstrated. The present study was undertaken to examine the effects of somatostatin on rat PRL secretion in several different circumstances where the circulating PRL level is elevated: (1) the estradiol primed intact male rat, (2) normal and (3) estradiol primed rats pretreated with pimozide, (4) normal and (5) estradiol primed hypophysectomized male rats with adenohypophyses grafted under the kidney capsule (HAG rat). Blood samples (70 microL) were taken every 2 min via an indwelling atrial cannula from conscious, unrestrained animals. In the estradiol primed intact rats, a bolus injection of somatostatin (10, 100, and 1000 micrograms/kg) lowered PRL levels in a dose-dependent manner. When the PRL concentration was elevated by the administration of pimozide (3 mg/kg), a dopaminergic receptor blocking agent, somatostatin was ineffective in decreasing plasma PRL concentration but the PRL concentration was lowered by somatostatin when the rat had been primed with estradiol. Somatostatin had no effect on the normal HAG rats, but lowered the plasma PRL concentration in the estradiol primed HAG rats. Since somatostatin inhibits PRL secretion only in the estradiol primed rats, it is suggested that estradiol priming creates a new environment, presumably via new or altered receptors, which can be inhibited by somatostatin.     

Somatostatin stimulates ductal bile absorption and inhibits ductal bile secretion in mice via SSTR2 on cholangiocytes

With an in vitro model using enclosedintrahepatic bile duct units (IBDUs) isolated from wild-type andsomatostatin receptor (SSTR) subtype 2 knockout mice, we tested theeffects of somatostatin, secretin, and a selective SSTR2 agonist(L-779976) on fluid movement across the bile duct epithelial celllayer. By RT-PCR, four of five known subtypes of SSTRs (SSTR1,SSTR2A/2B, SSTR3, and SSTR4, but not SSTR5) were detected incholangiocytes in wild-type mice. In contrast, SSTR2A/2B werecompletely depleted in the SSTR2 knockout mice whereas SSTR1, SSTR3 andSSTR4 were expressed in these cholangiocytes. Somatostatin induced adecrease of luminal area of IBDUs isolated from wild-type mice,reflecting net fluid absorption; L-779976 also induced a comparabledecrease of luminal area. No significant decrease of luminal area byeither somatostatin or L-779976 was observed in IBDUs from SSTR2knockout mice. Secretin, a choleretic hormone, induced a significantincrease of luminal area of IBDUs of wild-type mice, reflecting netfluid secretion; somatostatin and L-779976 inhibited (P < 0.01) secretin-induced fluid secretion. The inhibitory effect ofboth somatostatin and L-779976 on secretin-induced IBDUsecretion was absent in IBDUs of SSTR2 knockout mice. Somatostatin induced an increase of intracellular cGMP and inhibitedsecretin-stimulated cAMP synthesis in cholangiocytes; depletion ofSSTR2 blocked these effects of somatostatin. These data suggest thatsomatostatin regulates ductal bile formation in mice not only byinhibition of ductal fluid secretion but also by stimulation of ductalfluid absorption via interacting with SSTR2 on cholangiocytes, aprocess involving the intracellular cAMP/cGMP second messengers.

     

NMDA inhibits oxotremorine-induced acid secretion via the NO-dependent cyclic GMP system in rat stomach

The mechanism of N-methyl-D-aspartate (NMDA) inhibits oxotremorine-induced acid secretion was examined in rat stomach, in relation to the cyclic GMP system. NMDA (10(-7) M) did not affect the spontaneous acid secretion from the everted preparations of isolated rat stomach, but inhibited the acid secretion stimulated by oxotremorine, and this effect of NMDA was antagonized by 2-amino-5-phosphonovaleric acid (AP-5), (+/-)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) or N(G)-nitro-L-arginine (L-NNA). NMDA also elevated the cyclic GMP content of mucosal slices from rat stomach, and this effect of NMDA was antagonized by L-NNA. These results indicate that NMDA receptors are present in the rat stomach and regulate the gastric acid secretion. The mechanism underlying the effect of NMDA inhibits oxotremorine-induced acid secretion may be mediated by the NO-dependent cyclic GMP system.     

Phosphatidyl inositol inhibition of a sperm cyclic AMP-independent protein kinase

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei.

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.     

Psicose inhibits lettuce root growth via a hexokinase-independent pathway

Fructose analog, psicose, and glucose analog, mannose, inhibited root growth of lettuce seedlings. Psicose is phosphorylated by hexokinase and fructokinase (EC 2.7.1.4) to psicose-6-phosphate with no known capacity for further metabolism. Mannose is phosphorylated by hexokinase (EC 2.7.1.1) to mannose-6-phosphate which is further metabolized very slowly. Hexokinase is known to have a sugar-sensing function and possibly triggers a signal cascade resulting in changes of several gene expressions. It was determined, compared with the behaviour of mannose, whether psicose inhibits the root growth through this system. The addition of phosphate into the growth medium of lettuce seedlings did not affect the inhibition by psicose and mannose, and both sugars did not reduce adenosine triphosphate (ATP) level in the roots, suggesting that the inhibition is not due to phosphate starvation and ATP depletion. The inhibiting effects of psicose and mannose were overcome by adding sucrose into the medium, which suggests that the inhibition is not caused by accumulation of psicose-6-phosphate or mannose-6-phosphate in the seedlings. Mannoheptulose, a specific competitive inhibitor of hexokinase, defeated the mannose-induced inhibiting but was not able to relieve the psicose-induced inhibition. Thus, the phosphorylation of mannose by hexokinase may trigger a signal cascade resulting in the growth inhibition of lettuce roots, which is consistent with the hypothesis established in Arabidopsis . However, psicose cannot inhibit the growth of lettuce roots via a hexokinase-mediated pathway, and the phosphorylation of psicose by fructokinase might trigger a hexokinase-independent signal cascade resulting in the growth inhibition.     

Somatostatin inhibits VIP- and isoproterenol-stimulated cyclic AMP accumulation in rat prostatic epithelial cells

The dual regulation of cyclic AMP accumulation was studied in rat prostatic epithelial cells incubated with somatostatin, vasoactive intestinal peptide (VIP), and the beta-adrenergic agent isoproterenol. Somatostatin noncompetitively inhibited the stimulatory effect of VIP and isoproterenol, but it did not alter basal cyclic AMP levels. In addition to the multifactorial regulation of the cyclic AMP system in rat prostatic epithelium, these results suggest that somatostatin may play a physiological role at this level.     

Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP-independent pathway in osteoblastic clone MC3T3-E1 cells

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.     

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.     

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.     

Heterologous desensitization of the cyclic AMP-independent glycogenolytic response in rat liver cells.

Vasopressin and alpha-adrenergic agonists are known to be potent cyclic AMP-independent Ca2+-dependent activators of liver glycogen phosphorylase. When hepatocytes are pre-incubated with increasing concentrations of vasopressin or of the alpha-agonist phenylephrine, they become progressively unresponsive to a second addition of the respective agonist. The relative abilities of six vasopressin analogues and of five alpha-agonists to activate glycogen phosphorylase and to cause subsequent desensitization are highly correlated, indicating that the same vasopressin and alpha-adrenergic receptors are involved in both responses. About 5-times-higher peptide concentrations are needed to desensitize the cells than to activate their glycogen phosphorylase, whereas the concentrations of alpha-agonists required for the desensitization are only twice those needed for the activation of phosphorylase. The desensitization is not mediated by a perturbation in the agonist-receptor interaction. It is clearly heterologous, i.e. it is not agonist-specific, and must therefore involve a mechanism common to both series of agonists. The evidence for a role of Ca2+ movements or phosphatidylinositol turnover is briefly discussed.