Levels of brain cyclic AMP and cyclic GMP during the initiation of adult development in the bertha armyworm,Mamestra configurata Wlk

Levels of brain cyclic AMP and cyclic GMP in Mamestra configurata remained essentially unchanged during the first three days of pharate adult development after pupae were transferred from eight months of cold storage at 5° to 20°C. Thereafter, cyclic AMP levels increased six-fold over a two week period from 0.22 pmole/brain to about 1.3 pmole/brain. Cyclic GMP levels doubled over the two week period, from 0.09 pmole/brain to about 0.19 pmole/brain. These changes appeared to follow the activation of brain neurosecretion rather than precede it.Brain cyclic AMP and cyclic GMP levels of non-developing pupae remained virtually unchanged at about 0.27 and 0.048 pmole/brain, respectively, following their transfer from two months of cold storage at 5° to 20°C. Cyclic AMP: cyclic GMP ratiosthus increase from about 2:1 in the pupal brain to 6:1 oreven 7:1 in the differentiating brain of the pharate adult.The results are consistent with an important role for cyclic AMP and cyclic GMP in brain processes that become established during differentiation of the adult brain, and perhaps in the case of cyclic AMP, in the differentiation process itself.     

Cyclic AMP and cyclic GMP in hyperresponsiveness

Cyclic-AMP has been shown to cause a hyperresponse in blood pressure change in conjunction with norepinephrine in the anesthetized rat system. Recent experiments show that the antagonist to angiotensin II, Sar1-Thr8 angiotensin II, abolishes the hyperresponse produced by c-AMP. This is interpreted to mean that the added response caused by c-AMP is mediated through angiotensin II. When the antagonist is removed, the hyperresponse is observed to return. The experiments with cyclic-GMP indicate that the hyperresponse seen with c-AMP is not only absent, but a constant decrease in response to norepinephrine is observed as long as c-GMP is present. The decrease in blood pressure change in the presence of c-GMP suggests that the 10-5M c-GMP causes a relaxation of vascular smooth muscle. These two cyclic nucleotides seem to produce their effects by two completely different mechanisms.     

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10⁻¹² to 10⁻⁸ mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10⁻¹⁰ mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD⁺ measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.     

Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum

The subcellular localization of cyclic GMP and cyclic AMP in the rat caudate-putamen has been studied using horseradish peroxidase immunocytochemistry. Both of the putative neurotransmitter second messengers were visualized in neurons and glial cells at light microscopic resolutions, but not all cells of either category gave detectable staining. This was confirmed at the ultrastructural level where both stained and unstained elements of the same cell type were found within the same field. A striking variation was seen in cyclic nucleotide staining intensity within individual neural and glial cells. Both of the cyclic nucleotides were detected within postsynaptic terminal boutons and within astroglial processes. Cyclic GMP postsynaptic staining was stronger than glial staining, whereas the localization pattern was reversed for cyclic AMP. The synaptic localization of cyclic AMP and cyclic GMP immunoreactivity adds support to the idea that these compounds have an influential role in synaptic function within the striatum.     

Rapid assay for cyclic AMP and cyclic GMP phosphodiesterases.

A rapid highly sensitive assay for cyclic AMP phosphodiesterase has been devised. After a 5-min incubation, cyclic AMP is readily resolved from 5′-AMP, adenosine, and inosine by ion-exchange thin-layer chromatography on 1.3 × 6.5-cm strips of PEI-cellulose for 7 to 8 min. This procedure combines the accuracy of the standard paper chromatography assay (1) with the speed of ion-exchange resin techniques (2), while surmounting some of the major drawbacks of the other two methods (3). Since chromatography on PEI-cellulose efficiently resolves cyclic GMP, 5′-GMP, and guanosine, this methodology has also been adapted to the measurement of cyclic GMP hydrolysis.     

Cyclic AMP and cyclic GMP in rabbit blastocysts.

Concentrations of both nucleotides were significantly higher in Day-6 than in Day-5 blastocysts but the ratio of cAMP to cGMP changed from 0.5 to 1.5.     

Simultaneous quantification of GMP, AMP, cyclic GMP and cyclic AMP by liquid chromatography coupled to tandem mass spectrometry

Phosphodiesterases are drug targets for treating various diseases. Inhibition of these can increase cAMP and cGMP levels, which can affect a variety of physiological responses. Here we report a new method for determining PDE activity by combining high-performance liquid chromatography and tandem mass spectrometry. Characteristic peaks of the substrates, cGMP or cAMP and products, GMP or AMP, were identified in positive-ion electrospray ionization using multiple reaction monitoring. The method can be applied to determine activity of PDE inhibitors. Our results showed that this new method was fast, sensitive and highly reproducible.     

Specific binding proteins for cyclic AMP and cyclic GMP in Dictyostelium discoideum.

In Dictyostelium discoideum both cyclic AMP and cyclic GMP are regulated by chemotactic stimuli. Binding proteins specific for cAMP and cGMP have been found in aggregation competent cells as well as in cells harvested during growth. The activity of binding proteins was, on the average, lower in the growth phase cells. cAMP binding proteins were separated into 3 fractions, whereas the cGMP binding activity appeared in 1 major peak both on DEAE-cellulose and Sephadex G-200. Protein kinase activity was present in most but not all cyclic necleotide binding fractions; evidence for a relationship is however missing.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts

Polyacrylamide slab gel electrophoresis and [35S]methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.     

Stump currents in regenerating salamanders and newts

We report here that a variety of salamanders and newts from differing habitats all drive a steady ionic electric current out of the forelimb stump tip after forelimb amputation. Several hours after amputation the density of this stump current ranges from about 10 to 100 microA/cm2 in most species, and declines with time. In most cases, the magnitude of the stump current is dependent on the concentration of Na+ in the external medium (an artificial pondwater), suggesting that the well-known Na+ -dependent transcutaneous voltage described in amphibia (particularly frogs) is the EMF for this stump current. These measurements add to those previously reported for the North American red spotted newt (Notophthalmus viridescens), and suggest that electrical changes following amputation of urodele limbs are widespread among members of this group.