Effect of clonidine on second messenger systems in rat adrenal gland.

Clonidine, an alpha 2-adrenergic agonist, also binds to non-adrenergic imidazole receptors in brain and peripheral tissues. In adrenal medulla, however, clonidine appears to bind only to imidazole receptors. To assess whether the signal transduction mechanism of imidazole receptors differs from alpha 2-adrenergic receptors, we studied the actions of clonidine on the turnover of phosphoinositide and the production of cAMP and cGMP in slices of rat adrenal gland. Clonidine did not modify basal or carbachol mediated increases in phosphoinositide turnover or production of cAMP, however it increased the production of cGMP. The increase in cGMP was slow and unaffected by the addition of the phosphodiesterase inhibitor, IBMX. We conclude that the second messenger response triggered by clonidine in adrenal differs from that usually coupled to alpha 2-adrenergic receptors. Whether the effect is mediated by cell surface imidazole receptors remains to be established.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue.

The intracellular level of cGMP was independent of the rate of cell division in cells derived from virally infected brain tissue. The phosphodiesterase inhibitor R07-2956 (4-dimethoxybenzyl-2-imidazolidinone) increased the intracellular level of cGMP in virally infected brain cells, but it did not effect the level of cAMP. There was no correction between the increase in cGMP levels following addition of R07-2956 and changes in mitotic activity in the brain cell cultures. Experimental manipulations which increased the cAMP level were accompanied by a decreased mitotic rate indicating there was a correlation between mitotic activity and the level of cAMP in the same cells. Raising the intracellular level of cAMP by exogenous db-cAMP or cAMP or the use of other phosphodiesterase inhibitors routinely increased the level of cGMP as well. Conversely increasing the intracellular cGMP level by adding the exogenous cGMP increased the level of both cGMP and cAMP.A tissue culture system was used with the cell line derived from viral infected human brain tissue originally obtained from a patient with subacute sclerosing panencephalitis (SSPE). The intracellular levels of cAMP and cGMP were monitored by radioimmunoassay following manipulation of the system by addition of exogenous cGMP (0.05 mM), addition of exogenous db-cAMP (0.5 mM), or cAMP (0.5 mM) and the use of phosphodiesterase inhibitors: theophylline (1.0 mM), papaverine (50 μg/ml), 4-3-butoxy-4-methoxy benzyl-2-imidozalidinone (R020-1724) and R07-2956. Cell division was monitored in treated and non-treated cultures at 24 h intervals by analyzing the cell number and mitotic index.High levels of cGMP were found in cells which were not actively dividing but high levels were just as apt to be present in dividing cells. There was an inverse relationship between cell division and the level of cAMP.     

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

Comparison of the muscarinic-cholinergic and lysophosphatidate inhibition of fibroblast adenylate cyclase demonstrating desensitization to the cholinergic stimulus

Muscarinic stimulation of cultured fibroblasts decreases initial rates of cAMP accumulation in response to hormones 50–70%. This inhibitory effect of muscarinic stimulation on cAMP accumulation in intact cells was desensitized 65–75% by a 60 min pretreatment with the muscarinic agonist carbachol (10 μM), with a $\text{t}\text{1}\text{2}$ of 11 min. The carbachol pretreatment resulted in a diminished carbachol inhibition of adenylate cyclase in broken cell preparations. The phospholipid monooleylphosphatidate (MOPA) which also inhibited hormone-stimulated cAMP accumulation with a half maximal effect at 0.03 μM (as compared with 0.5 μM for carbachol), displayed many of the characteristics of muscarinic inhibition such as loss of activity with time of pretreatment. However, fibroblasts did not become desensitized to prolonged MOPA treatment; rather, it appeared that the MOPA was being inactivated. Also, the desensitization to carbachol did not prevent further inhibition by MOPA. The inhibitory effects of maximal doses of MOPA and carbachol in combination were no greater than the effect of carbachol alone, suggesting that they shared an intermediate in their inhibition of cAMP accumulation. These results are consistent with the hypothesis that muscarinic inhibition of adenylate cyclase is mediated by the formation of a phospholipid. However, the desensitization to the cholinergic stimulus does not appear to involve the intermediate, but rather a modification at the receptor level.     

Cyclic GMP and cyclic AMP induced changes in control and hypertrophic cardiac myocyte function interact through cyclic GMP affected cyclic-AMP phosphodiesterases.

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.     

Neurohormonal receptors and cyclic AMP-binding proteins in rabbit tracheal mucosa-submucosa

Neurohormones and drugs that alter in vitro tracheal electrolyte transport and mucus glycoprotein secretion were examined for their ability to alter cyclic nucleotide accumulation in a smooth muscle-free preparation of rabbit tracheal mucosa-submucosa. cAMP levels were increased by beta-adrenergic agonists, histamine, 2-Cl-adenosine and prostaglandin E1. cGMP levels were increased by carbachol. The phosphodiesterase inhibitor isobutylmethylxanthine increased cAMP and cGMP levels and potentiated only the beta-adrenergic effects. The beta-adrenergic effects were blocked by (+/-)-propranolol and the effects of histamine by diphenhydramine, atropine and (+/-)-propranolol. Atropine blocked the carbachol effects. The isolated surface epithelium from rabbit trachea had higher basal cAMP levels and greater response to beta-adrenergic agonists and isobutylmethylxanthine than the mucosa-submucosa. Two major cAMP-binding proteins in the tracheal mucosa-submucosa were identified with the photoaffinity label 8-N3-[32P]cAMP. Agents that increased cAMP levels also decreased photoaffinity labelling, suggesting that these two cAMP-binding proteins were being occupied in the intact cell. The molecular weights of the proteins were 50 000 and 54 000 and correspond in electrophoretic mobility to the regulatory subunits of Type-I and Type-II cAMP-dependent protein kinases, respectively. The results are consistent with the hypothesis that epithelial functions in the airways are modulated by a number of agonists which increase cyclic nucleotide levels. The effects of beta-adrenergic agonists is apparently mediated by activation of adenylate cyclase and subsequent activation of cAMP-dependent protein kinases.     

The effects of valproate and phenytoin on the cAMP and cGMP levels in nervous tissue

Earlier studies have demonstrated that valproic acid (VPA) and phenytoin (PHT) influence the excitability properties of crayfish axons through different mechanisms. PHT was found to antagonize the electrophysiologic effects of VPA. The purpose of the present study was to determine if the electrophysiologic effects of VPA and PHT are correlated with changes in the cellular levels of either cAMP or cGMP as these substances are known to influence membrane excitability. It was found that PHT (0.1 mM) has no effect on the levels of either cAMP or cGMP within crayfish neural tissue. VPA (4.0 mM) also has no effect on cAMP levels. However, it does significantly reduce the levels of cGMP. Pretreatment of neural tissue with PHT has been shown to eliminate the effects of VPA on membrane excitability. It was found that this pretreatment has no influence on VPA's ability to reduce cGMP levels. The effect of VPA on cGMP levels is observed in the absence of spontaneous activity. Therefore, it is concluded that the observed reduction in cGMP levels does not represent the modulation of cGMP levels that is known to accompany activity. Two experiments demonstrate that the 4-mV depolarization of membranes by VPA can not account for its effect on cGMP levels. In the first, pretreatment with PHT abolished the depolarizing effect on VPA but not its effect on cGMP. In the second, a concentration of ouabain which depolarizes crayfish neural tissue by 8-10 mV without producing spike activity had no effect on either cAMP or cGMP levels. These experiments effectively dissociate the electrophysiologic response to VPA and PHT from changes in cyclic nucleotide levels.     

cGMP enhances the sonic hedgehog response in neural plate cells.

The elaboration of distinct cell types during development is dependent on a small number of inductive molecules. Among these inducers is Sonic hedgehog (Shh), which, in combination with other factors, patterns the dorsoventral (DV) axis of the nervous system. The response of a cell is dependent in part on its complement of cyclic nucleotides. cAMP antagonizes Shh signaling, and we examined the influence of cGMP on the Shh response. Cells in chick neural plate explants respond to Shh by differentiating into ventral neural-cell types. Exposure of intermediate-zone explants to cGMP analogs enhanced their response to Shh in a dose-dependent manner. The Shh response was also enhanced in dorsal-zone explants exposed to chick natriuretic peptide (chNP), which stimulates cGMP production by membrane-bound guanylate cyclase (mGC). Addition of chNP to intermediate-zone explants did not enhance the Shh response, consistent with a reported lack of mGC in this region of the neural tube. Finally, the presence of a nitric oxide (NO)-sensitive guanylate cyclase (GC) was established by demonstrating cGMP immunoreactivity in neural tissue following NO stimulation of whole chick embryos. Intracellular levels of cGMP and cAMP may thus provide a mechanism through which other factors modulate the Shh response during neural development.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Cross-Regulation of Intracellular cGMP and cAMP in Cultured Human Corpus Cavernosum Smooth Muscle Cells

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 μM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 μM of forskolin or PGE₁ produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K_i of 258 ± 54 μM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.     

Effect of radiation on cAMP,cGMP and Ca(2+)(i) pathways and their interactions in rat distal colon

The secretory response implicated in the intestinal response to luminal attack is altered by radiation. The cAMP, cGMP and Ca(2+)(i) pathways leading to secretion as well as the interactions between the cAMP pathway and the cGMP or Ca(2+)(i) pathway were studied in the rat distal colon 4 days after a 9-Gy abdominal X irradiation, when modifications mainly occurred. The secretory response in Ussing chambers and cAMP and cGMP accumulation in single isolated crypts were measured. The muscarinic receptor characteristics were determined in mucosal membrane preparations. The secretory response by the cAMP pathway (stimulated by vasoactive intestinal peptide or forskolin) and the cAMP accumulation in crypts were decreased (P < 0.05) after irradiation. The weak secretory response induced by the cGMP pathway (stimulated by nitric oxide or guanylin) was unaltered by radiation, and the small amount of cGMP determined in isolated crypts from the control group became undetectable in the irradiated group. Inducible NOS was not involved in the hyporesponsiveness to VIP after irradiation (there was no effect of an iNOS inhibitor). The secretory response by the Ca(2+)(i) pathway (stimulated by carbachol) was unaffected despite a decreased number and increased affinity of muscarinic receptors. The non-additivity of VIP and carbachol co-stimulated responses was unmodified. In contrast, VIP and SNP co-stimulation showed that NO enhanced the radiation-induced hyporesponsiveness to VIP through a reduced accumulation of cAMP in crypts. This study provides further understanding of the effect of ionizing radiation on the intracellular signaling pathways.     

Fluoride inhibits agonist-induced formation of inositol phosphates in rat cortex

Sodium fluoride inhibited carbachol, 5-hydroxytryptamine and noradrenaline stimulated formation of inositol phosphates in rat cerebral cortex. For example, carbachol (1 mM) induced a 337% increase of inositol phosphates above basal in 30 min which was reduced to 69% in the presence of NaF (10 mM). The IC50 for NaF was approximately 1.5 mM and inhibition was mediated by a decrease in maxima of the carbachol dose response curve rather than a shift to the right. This inhibitory action was not mimicked by NaBr or NaI, or by agents which increase cAMP. Inhibition did not appear to result from a toxic action of NaF since it had no effect on the formation of inositol phosphates by high K+; moreover, in higher concentrations NaF stimulated phospholipase C activity. Since fluoride ions are known to activate G-proteins in the concentrations used in this study, these results may indicate the existence of a novel G-protein linked to receptor inhibition of phospholipase C.     

Regulation of cytosolic calcium by cAMP and cGMP in freshly isolated smooth muscle cells from bovine trachea

The regulation of the cytosolic calcium concentration was investigated in freshly isolated adult bovine tracheal smooth muscle cells using fura 2. These cells contain 1.1 and 1.8 pmol of cGMP kinase and cAMP kinase per mg protein, respectively. Carbachol, histamine, serotonin, isoproterenol, and salbutamol increased the cytosolic calcium in a dose-dependent manner from 79 nM to about 650 nM. Preincubation of these cells for 20 min with isoproterenol, forskolin, 8-Br-cAMP and 8-(4-Cl-phenyl)thio-cAMP did not lower carbachol-induced increases in cytosolic calcium concentration, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the atrionatriuretic factor, isobutylmethylxanthine, and 8-Br-cGMP lowered cytosolic calcium. The active fragment of cGMP kinase, but not the catalytic subunit of cAMP kinase lowered carbachol-induced calcium levels. Carbachol released calcium from intracellular stores and increased calcium influx from the extracellular space. The influx was inhibited by preincubation with the calcium channel blockers nitrendipine or gallopamil. Both carbachol-stimulated pathways were suppressed by 8-Br-cGMP. Isoproterenol increased only the influx of calcium from the outside by a channel which was blocked by calcium channel blockers or 8-Br-cGMP. Forskolin and 8-Br-cAMP lowered carbachol- and isoproterenol-stimulated increases in calcium when added shortly before or after the addition of the agonist. In addition, isoproterenol decreased carbachol-stimulated calcium levels when added 10 s after carbachol. The calcium stimulatory effect of isoproterenol was abolished by preincubation of the cells with pertussis toxin or cholera toxin. These results show (a) that the beta 2-adrenoceptor couples in isolated tracheal smooth muscle cells to a dihydropyridine- and pertussis toxin-sensitive calcium channel; (b) that the same channel is opened by carbachol; (c) that cGMP kinase is very effective in decreasing elevated cytosolic calcium concentrations, whereas cAMP-dependent protein kinase has a variable effect on stimulated cytosolic calcium levels.     

Diverse directional changes of cGMP relative to cAMP in E. coli.

The relationships of the changes of cAMP and cGMP concentrations in $\text{E. coli}$ varied as a function of experimental conditions. (1) Cells starved for carbon source for a short time period had high cAMP and low cGMP concentrations. Addition of carbon source (succinate, glucose or α-methyl glucoside) led to a decrease in cAMP and an increase in cGMP (bi-directional change). (2) Washed cells starved for glucose for long time periods had low cAMP levels which did not change on glucose addition. Addition of succinate or glucose to such cells led to a transient increase in cGMP levels (uncoupled change). The cGMP concentration peaked at 15 minutes or 1 hour after glucose or succinate addition, respectively. (3) Sham shift-up experiments (addition of α-methyl glucoside to cultures growing in succinate) in $\text{E. coli}$ 1100 and CA 8000 showed decreases in cGMP levels in both strains; however, cAMP levels increased in the former (bi-directional change) and decreased in the latter (unidirectional change).     

Changes in the cyclic nucleotides of rat thyroid, pituitary and plasma caused by methylthiouracil treatment.

Changes in the content of cyclic nucleotides (cAMP and cGMP) and related enzyme activities were observed in the rat thyroid, pituitary and plasma during the prolonged increase of endogenous TSH produced by treatment with methylthiouracil (MTU). Experiments were performed after 4 weeks treatment with MTU. The wet weight and cAMP content per wet weight of the thyroid increased 3 and 1.4 times respectively, but cGMP showed a slight decrease. Pituitary weight increased 1.3 times, but cAMP and cGMP content did not change. The cAMP level in plasma also increased about 1.3 times, but cGMP did not increase. The cAMP-phosphodiesterase activity in the thyroid, pituitary and plasma was increased 1.9, 1.4 and 1.3 times respectively after MTU treatment, while cGMP-phosphodiesterase showed no significant change. ATPase activity in the thyroid and pituitary was also increased more than 1.5 times after MTU treatment, while 5'-nucleotidase activitity decreased remarkably. These data indicate that the metabolism of the cyclic nucleotide system in the thyroid is stimulated by TSH.