Forskolin- and dibutyryl cyclic AMP-mediated inhibition of chondrogenesis.

The regulatory role of cyclic AMP in various cellular activities is well known. It has been documented that both the notochord and extracellular matrix materials (ECM) induce somite chrondrogenesis. We believe that the ECM modulates the intracellular cAMP level during chondrogenic differentiation. The studies indicated that notochordal induction, which resulted in somite chondrogenesis (reflected by increased sulfated glycosaminoglycan synthesis) reduced the intracellular cAMP level in somites. Addition of forskolin and dibutyryl cAMP resulted in increased intracellular cAMP levels and decreased synthesis of sulfated glycosaminoglycans (decreased chondrogenesis). In the case of dibutyryl cAMP, the inhibition of sulfated glycosaminoglycan synthesis was related to the length of exposure time. Thus, the inverse relationship between cAMP content and enhanced chondrogenesis supports the theory that, in somites, a decrease in the intracellular cAMP level may be necessary to trigger chondrogenic differentiation.

Adrenergic transmitter release.

Presynaptic release-modulating receptors on sympathetic nerve fibres have been found not only in a great number of animal tissues but also in various human blood vessels. Although basically the same types of receptors are present in animals and man (e.g., inhibitory muscarinic receptors and alpha 2-adrenoceptors; facilitatory beta-adrenoceptors), there may be considerable species differences if a certain blood vessel or the effect of a certain agonist or antagonist is considered. Hormones may also influence noradrenergic transmission by acting presynaptically. Thus, ACTH increases the impulse-evoked noradrenaline (NA) release in the rabbit pulmonary artery by activating presynaptic ACTH receptors, and an adenylate cyclase may be involved in this effect. Results obtained with new pharmacological tools (forskolin and selective inhibitors of cAMP phosphodiesterase) support the suggestion that the sympathetic nerves of the rabbit pulmonary arteries are endowed with an adenylate cyclase activation of which increases evoked NA release.

Enhanced killing effects of caffein post-treatment in ultraviolet-light irradiated mouse lymphoma cells: is cAMP a mediator of the effects?

Effects of post-treatment with caffein, cyclic adenosine 3':5'-monophosphate (cAMP) and N6, O2-dibutyryl cAMP (dbcAMP) were investigated in ultraviolet light (UV)-irradiated mouse lymphoma L5178Y cells. Under conditions where UV or each chemical alone caused only slight cytotoxic effects, caffein post-treatment showed clear synergistic effects in cell killing but not for cAMP or dbcAMP. Subsequently, a mutant clone resistant to cAMP was isolated. This mutant was supposed to be deficient in cAMP-mediated cellular functions. Using the mutant cells, it was found that these cells were also sensitive to caffein post-treatment as wild cells after UV-irradiation. The results imply that the enhanced killing effects by caffein post-treatment in UV irradiated cells is not mediated by cAMP.

Nicotinamide restores phosphaturic effect of PTH and calcitonin in phosphate deprivation.

Results of previous studies suggest a potentially important role for nicotinamide adenine dinucleotide (NAD) in the cellular regulation of phosphate transport by the renal proximal tubules. The present clearance studies were performed to evaluate whether intraperitoneal administration of nicotinamide, a precursor of NAD and inhibitor of NAD catabolism, would not only increase phosphate excretion but also restore the phosphaturic response to parathyroid hormone (PTH) in rats fed a low phosphate diet. Rats fed a low phosphate diet were resistant to the phosphaturic effect of PTH, calcitonin, and dibutyryl cAMP (DBcAMP) in spite of the fact that all three agents elicited an increase in the urinary excretion of cAMP. Administration of nicotinamide to rats fed a low phosphate diet increased renal cortical NAD levels, increased phosphate excretion, partially restored the phosphaturic effect of PTH and DBcAMP, and completely restored the phosphaturic response to calcitonin. We conclude that nicotinamide restores the phosphaturic effect of PTH and calcitonin in rats fed a low phosphate diet by acting at a cellular step subsequent to cAMP generation to inhibit tubular reabsorption of phosphate.

Refractoriness of TSH- and PGE2-stimulated iodine metabolism in cultured porcine thyroid cells, evidence for refractoriness at the level of cAMP action.

In an attempt to study intrinsic regulatory mechanism involved in iodine metabolism, chronic and acute effects of TSH, PGE2 and DBC on iodine uptake, iodide discharge and organic binding of iodine were examined using cultured porcine thyroid cells. Culture in the presence of TSH, PGE2 and DBC for 6 days maintained the ability to thyroid cells to take up iodine and organify it, but culture in the absence of these substances failed to do so. When incubated with NaI in the presence of 1 mM methylmercaptoimidazole (MMI), the cells took up iodide and this accumulated iodide was discharged by TSH, pGE2 and DBC. TSH-, PGE2, and DBC-stimulated iodide discharge was depressed greatly after chronic exposure to TSH, PGE2 or DBC. This refractoriness of TSH-, PGE2- or DBC-stimulated iodide discharge was not specific for each thyroid stimulating substance; previous exposure to TSH, PGE2 or DBC induced refractoriness of TSH-, PGE2- and DBC-stimulated iodide discharge, providing evidence for the existence of refractoriness at the level of cyclic AMP action on iodide discharge. When incubated with NaI in the absence of MMI, the cells took up iodide and organified it. After 30 min incubation with NaI, TSH, PGE2 and DBC were added and they stimulated iodide organification further. This TSH- and PGE2-stimulated iodide organification was also depressed after exposure to TSH or PGE2. These data indicate that, as an intrinsic regulatory mechanism, refractoriness is operating at the level of cAMP action on iodine discharge and organification.

Interactions of starvation and selective phosphorus depletion on renal phosphate reabsorption.

Renal phosphate (Pi) wastage following 7 days of starvation was investigated in normal rats (HI-P) and others previously stabilized on a low phosphorus (LO-P)diet. In LO-P animals, Pi excretion increased after starvation, but was significantly less than in starved HI-P rats. After thyroparathyroidectomy, the increase in Pi excretion after parathyroid hormone (PTH) was significantly greater in nonacidotic starved HI-P rats than in LO-P animals. However, PTH elicited a 31-fold increase in Pi excretion in both of these groups. Starved LO-P and HI-P rats responded equivalently to dibutyryl cyclic AMP. The renal response to phosphate depletion normally promotes Pi conservation, but is attenuated markedly by 7 days of subsequent starvation. This results from at least partial restoration of phosphaturic responsiveness to PTH during starvation.

Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.

Hormonal release of programmed behavior in silk moths: probable mediation by cyclic AMP.

The eclosion hormone triggers a stereotyped preprogrammed pattern of behavior in silk moths. The effects of the hormone were duplicated by the injection of dibutyryl adenosine 3', 5'-monophosphate, adenosine 3', 5'-monophosphate (cyclic AMP), or guanosine 3', 5'-monophosphate (cyclic GMP) into theophylline-treated pharate moths. Treatment with theophylline reduced the latency of the response to a low dose of hormone, presumably by blocking phosphodiesterase. Endogenous levels of cyclic AMP, but not cyclic GMP, increased significantly in the central nervous system within 10 minutes after hormone injection. We conclude that an early step leading to the release of the eclosion motor program is an increase in cyclic AMP in target neurons of the central nervous system.

The effect of an inhibitor of adenylate cyclase on the development of pyrogen, prostaglandin and cyclic AMP fevers in the rabbit.

An exotoxin of Bacillus thuringiensis known to inhibit adenylate cyclase in vitro has been used to investigate the role of cyclic AMP in the pathogenesis of fever in the rabbit. Intra-hypothalamic microinjections of the exotoxin are non-pyrogenic and significantly attenuate the hyperthermia caused by intrahypothalamic microinjections of both bacterial pyrogen (endotoxin) and prostaglandin E1. The hyperthermia produced by dibutyrl cyclic AMP is not affected by the exotoxin. These results support the idea that adenylate cyclase is activated during the development of fever in the rabbit.

The role of renal adenosine 3',5'-monophosphate in the control of erythropoietin production.

A regulatory role for adenosine 3',5'-monophosphate (cyclic AMP) in the production of the renal hormone rythropoietin following erythropoietic stimulation with cobaltous chloride hexahydrate is proposed. Studies in rates reveal a temporal relationship between renal cyclic AMP levels and plasma titers of erythropoietin. In addition, cobalt increases the activity of an erythropoietin-generating enzyme (renal erythropoietic factor) with maximal enzyme activity occurring after the rise in cyclic AMP levels but before the increase in erythropoietin titers. This increase in renal cyclic AMP is localized to the renal cortex. Cobalt stimulates renal cortical adenylate cyclase but has no effect on renal cyclic nucleotide phosphodiesterase. The addition of cyclic AMP (3 time 10-6 M) and a partially purified cyclic AMP-dependent protein kinase from rat kidney to an inactive preparation of renal erythropoietic factor increases the ability of renal erythropoietic factor to generate erythropoietin. Data from the polycythemic mouse assay, a bioassay used to quantitate erythropoietic activity of test substances, indicate that dibutyryl cyclic AMP is erythropoietically active with respect to its ability to increase radioactive-labelled iron (59Fe) incorporation into heme of newly formed red blood cells. Theophylline, which by itself is erythropoietically inactive, potentiated the erythropoietic effect of cobalt in polycythemic mice. These results suggest that cyclic AMP plays a significant role in the renal production of erythropoietin following cobalt administration. It is postulated that cobalt stimulates renal cortical adenyoate cyclase, thus increasing renal cyclic AMP levels. Cyclic AMP then activates a protein kinase which subsequently stimulates renal erythropoietic factor to generate erythropoietin. A similar cyclic AMP mechanism may be operative after erythropoietic stimulation by exposure to hypoxia or prostaglandin treatment.