Specific Activity Of Adenylate Cyclase Research Articles

Evaluation of Cytotoxicity and Hypoxic Effect of Nitroimidazole Embedded Nanoparticles.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

Methylseleninic acid is a novel suppressor of aromatase expression

Elevated circulating estrogen levels, as a result of increased peripheral aromatization of androgens by aromatase, have been indicated to underlie the association between obesity and a higher risk of breast cancer in postmenopausal women. Although aromatase inhibitors have been used as a first-line therapy for estrogen receptor-positive breast cancer in postmenopausal women, their potential as breast cancer chemopreventive agents has been limited due to toxicities and high costs. It is therefore imperative to develop new aromatase-inhibiting/suppressing agents with lower toxicities and lower costs for breast cancer chemoprevention, especially in obese postmenopausal women. The expression of the aromatase gene, CYP19, is controlled in a tissue-specific manner by the alternate use of different promoters. In obese postmenopausal women, increased peripheral aromatase is primarily attributed to the activity of the glucocorticoid-stimulated promoter, PI.4, and the cAMP-stimulated promoter, PII. In the present study, we show that methylseleninic acid (MSA), a second-generation selenium compound, can effectively suppress aromatase activation by dexamethasone, a synthetic glucocorticoid, and forskolin, a specific activator of adenylate cyclase. Unlike the action of aromatase inhibitors, MSA suppression of aromatase activation is not mediated via direct inhibition of aromatase enzymatic activity. Rather, it is attributable to a marked downregulation of promoters PI.4- and PII-specific aromatase mRNA expression, and thereby a reduction of aromatase protein. Considering the low-cost and low-toxicity nature of MSA, our findings provide a strong rationale for the further development of MSA as a breast cancer chemopreventive agent for obese postmenopausal women.

Effect of Stimulators upon Human Hepatic Adenylate Cyclase Activity: A Tool of Nitroimidazole Cytotoxicity Assessment

AbstractNitroimidazole is an antibiotic and radiosensitizer chemical with great potentials in imaging. The hepato-cytoxicity evaluation of nitroimidazole and possible energy status changes in liver cells due to its anti-inflammatory characteristics are crucial in tumor imaging and chemotherapy. Adenylate cyclase is key enzyme to cause energy imbalance leading to cytotoxicity. To evaluate energy status in hepatocytes and Kupffer cells, adenylate cyclase activities in isolated liver cells were compared in presence of stimulators. To evaluate the effect of effectors on adenylate cyclase in cultured hepatocyte cells, adenylate cyclase enzyme was stimulated by different GITP, GTP, progesterone and nitroimidazole effectors. In cultured Kupffer cells, prostaglandin E~2~ and F~2[alpha]~ were used as effectors. The results showed that nitroimidazole decreased adenylate cyclase specific activity in dose-dependent manner after pre-incubation of hepatocytes with the nitroimidazole in medium. Nitroimidazole stimulated adenylate cyclase activities in hepatocytes were mediated by cAMP and determined by cAMP mesurement. The stimulatory effect of nitroimidazole on adenylate cyclase was independent of the GTP presence in the assay system perhaps due to a direct effect on the catalytic subunit of adenylate cyclase enzyme. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the nitroimidazole. In conclusion, nitroimidazole exhibits direct effect on the catalytic subunit of the adenylate cyclase system. The adenylate cyclase was hormone sensitive in liver cells.

Cholera toxin induced gene expression alterations

The cholera toxin (CT) is a well-known inducer of cAMP and cAMP regulates gene expression of many genes. However, little is known as to the alterations in gene expression in response to CT. Here the alterations of the expression of 800 selected genes in response to CT were examined using cDNA microarrays. Gene expression alterations in human lymphocytes and monocytes were found after exposure to CT at varying concentrations for different time periods. Over 200 genes showed varying degrees of alterations of expression in CT-treated cells. The CT-induced changes in gene expression were compared by cDNA microarrays under the same conditions to those in response to forskolin, a specific activator of adenylate cyclase, and MDL-12, an irreversible inhibitor of adenylate cyclase. Thirty-five CT-responsive genes were found responded similarly to forskolin but differently to MDL-12. Fourteen CT-responsive genes were affected similarly by MDL-12 but differently by forskolin. Many of these CT responsive genes were involved in immunity, inflammation and oxidative stress. The CT induced responses correlated with those induced by CT subunits. The down regulation of Th1 markers and upregulation of Th2 markers by CT are consistent with the CT induction of Th2 cells.

5-HT(7)-like receptors mediate serotonergic modulation of photo-responsiveness of the medulla bilateral neurons in the cricket, Gryllus bimaculatus.

Serotonin (5-HT) suppresses the photo-responsiveness of medulla bilateral neurons (MBNs) that are involved in the coupling mechanism of the bilaterally paired optic lobe circadian pacemakers in the cricket, Gryllus bimaculatus. We found that forskolin, a highly specific activator of adenylate cyclase, mimicked the effects of serotonin on the MBNs. This fact suggests the involvement of cyclic 3', 5'-adenosine monophosphate (cAMP) in mediating the action of serotonin. We therefore tested the effects of various 5-HT receptor agonists and antagonists that are coupled to adenylate cyclase to specify the receptor involved. Application of 8-OH-DPAT that has affinity for both 5-HT(1A) and 5-HT(7) receptors suppressed the photo-responsiveness, like forskolin. The inhibitory effect of 8-OH-DPAT was effectively blocked by clozapine, a high affinity 5-HT(7) receptor antagonists with a very low affinity for 5-HT(2). Ketanserin, a selective 5-HT(2) antagonist, and NAN-190, a 5-HT(1A) antagonist, did not block it. These results suggest that serotonergic suppression of the photo-responsiveness of the MBNs is mediated by 5-HT(7)-like receptor subtypes.

GLUCAGON DECREASES CYTOKINE INDUCTION OF NITRIC OXIDE SYNTHASE AND ACTION ON INSULIN SECRETION IN RIN5F CELLS AND RAT AND HUMAN ISLETS OF LANGERHANS

Nitric oxide synthase, induced by cytokines in insulin-containing cells, produces nitric oxide which inhibits function and may promote cell killing. Since glucagon was shown to prevent inducible nitric oxide synthase (iNOS) expression in rat hepatocytes it was of interest to examine the action of glucagon (and cyclic AMP) on iNOS induction in insulin-producing cells. Cultured RIN5F cells and primary rat and human islets of Langerhans were treated with interleukin 1β (IL-1β) or a combination of cytokines, and were co-treated or pre-treated with glucagon. In RIN5F cells, the activity of iNOS induced by IL-1β (10pM, 24h), was significantly reduced by glucagon (1000nM), which raises cyclic AMP, and by forskolin (1–10μM), a non specific activator of adenylate cyclase. Glucagon and forskolin also decreased iNOS expression in RIN5F cells, and rat and human islets, as shown by Western blotting. The inhibitory action of IL-1β (100pM, 24h) on rat islet insulin secretion was partially reversed by 1-h pre-treatment with glucagon (10–1000nM), while the contrasting stimulatory effect of 48-h treatment with cytokines on insulin secretion from human islets was similarly prevented by glucagon (1000nM) pre-treatment. These results suggest that glucagon inhibits iNOS expression in insulin-containing cells and imply that glucagon could modulate the inhibitory effects of cytokines.

Activity trends of adenylate and guanylate cyclases and cAMP and cGMP levels during the first embryonic stages of Bufo bufo

1. 1. During the first stages of embryonic development of Bufo bufo, the levels of cAMP and cGMP showed interesting opposite trends analogous to the trends of their respective enzymes. 2. 2. In the late segmentation, the high increase of guanylate cyclase specific activity and the corresponding rise of embryonic level of cGMP, could indicate the involvement of this nucleotide in cellular proliferation. 3. 3. During the following stage of the gastrulation, the increase of adenylate cyclase specific activity, coupled to the loss of guanylate cyclase specific activity, could suggest the importance of cAMP in the phenomena of differentiation induction. 4. 4. Furthermore, the cytochemical investigation of adenylate and guanylate cyclase localization seems to confirm the prominent role of cAMP during the differentiation phases.

Effects of selected vasoactive substances on adenylate cyclase activity in brain, isolated brain microvessels, and primary cultures of brain microvessel endothelial cells.

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6 +/- 2.1, 15.6 +/- 2.7, and 8.4 +/- 1.5 pmol cAMP/mg protein/min +/- SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.

Ca2+ and cAMP activate different chloride efflux pathways in HT-29.cl19A colonic epithelial cell line.

The mechanism of adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-induced Cl- secretion was studied in monolayers of the colon carcinoma cell line HT-29.cl19A by combined short-circuit current (Isc) and 125I- or 36Cl- efflux measurements. Forskolin, a specific adenylate cyclase activator, was found to induce a large increase in Isc and a two- to threefold increase in 36Cl- efflux solely across the apical border. The fractional efflux of 36Cl-compared with 125I- (basal ratio 1.71 +/- 0.28) did not change significantly in the presence of forskolin (1.91 +/- 0.45). In contrast, the Ca2+ ionophore A23187 did not appreciably affect the Isc but enhanced 36Cl- and 125I- efflux at the apical and basolateral side of the monolayer. Furthermore, the fractional efflux ratio of 36Cl- to 125I- changed dramatically to a value of 0.36 +/- 0.14. Both forskolin- and A23187-induced 36Cl- or 125I- efflux were only weakly inhibited by the putative Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoicacid. Carbachol, a Ca(2+)-linked agonist, mimicked the effects of A23187 on the 36Cl- and 125I- efflux but additionally provoked a significant increase in Isc. These data show that Ca2+ and cAMP activate different Cl-efflux pathways in HT-29.cl19A cells. Most likely these pathways represent a cAMP-activated conductance in the apical membrane and a separate Ca(2+)-activated Cl- conductance expressed in both apical and basolateral membranes. Apparently cholinergic agonists induce net electrogenic Cl- secretion through an intracellular signaling pathway (e.g., protein kinase C activation) different from the one activated by Ca2+/Ca2+ ionophore alone.

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of α-subunits for G i-1 which were almost twice those in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the α-subunits of G i-2 and G i-3, and 42 and 45 kDa forms of G s and for G-protein β-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forsckolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE 1 and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of G 1-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.

Forskolin modulates acetylcholine release in the hippocampus independently of adenylate cyclase activation

[ 3H]Acetylcholine release from slices of rabbit hippocampus was elicited by electrical field stimulation (360 pulses/3 Hz). Both forskolin, commonly used as a specific activator of adenylate cyclase, as well as 1,9-dideoxy-for-skolin, which fails to activate adenylate cyclase, increased the evoked transmitter release in an almost identical manner. In addition, the phosphodiesterase inhibitor, rolipram, and the membrane-permeable analogue of cAMP, 8-Br-cAMP, did not influence acetylcholine release. These data show that forskolin is not specific to adenylate cyclase and that the increase in acetylcholine release in the rabbit hippocampus occurs through a mechanism other than activation of adenylate cyclase.

17β-Estradiol depolarization of hypothalamic neurons is mediated by cyclic AMP

The process by which 17β-estradiol rapidly modulates the excitability of neurons in the ventromedial hypothalamus, a facilitation center of female sexual behavior and satiety center of feeding behavior, through mediation by cyclic nucleotides, was investigated by intracellular recording from the guinea pig brain slice preparations. Two types of short-term responses were produced by depolarization with decreased K + conductance and hyperpolarization with increased K + conductance. These two responses were enhanced by the phosphodiesterase inhibitor, isobutylmethylxanthine. However, the specific adenylate cyclase activator, forskolin, enhanced only the depolarization. The analogue of cyclic adenosine 3′,5′-monophosphate (cAMP), 8-bromo-cAMP, induced only depolarization, the ionic mechanism of which was similar to that of 17β-estradiol. In addition, the possibility of non-specific effects of cyclic nucleotides was precluded by an experiment using an analogue of cyclic guanosine 3′,5′-monophosphate (cGMP), 8-bromo-cGMP, which hyperpolarized neurons. Thus, the present study strongly suggests that the production of depolarizing responses of neurons in the hypothalamus produced by estradiol is specifically mediated through cAMP.

Diabetes abolishes the GTP-dependent, but not the receptor-dependent inhibitory function of the inhibitory guanine-nucleotide-binding regulatory protein (Gi) on adipocyte adenylate cyclase activity

Adipocyte membranes from control rats exhibited a functional Gi (inhibitory guanine-nucleotide-binding protein) activity which could be assessed either by the inhibitory action of low concentrations of guanosine 5-[beta gamma-imido]triphosphate (p[NH]ppG) upon forskolin-stimulated adenylate cyclase activity or by the inhibitory action of high concentrations of GTP upon isoprenaline-stimulated adenylate cyclase activity. When membranes from animals made diabetic with streptozotocin were used, then both such inhibitory functions of Gi were abolished. In contrast, receptor-mediated inhibitory responses of Gi, effected by N6-phenylisopropyl (adenosine), prostaglandin E2 or nicotinate, were either unchanged or even apparently more effective in membranes from diabetic animals. Induction of diabetes did not cause any change in the adipocyte plasma membrane levels of the alpha, GTP-binding subunits of either Gi1 or Gi2 or of Gs (stimulatory guanine-nucleotide-binding protein), but elicited an increase in the level of alpha-Gi3. The induction of diabetes reduced the specific activity of adenylate cyclase in adipocyte membranes and enhanced the stimulatory effect of isoprenaline. It is suggested that diabetes causes selective changes in the functioning of Gi in adipocyte membranes which removes the tonic GTP-dependent inhibitory function of this G-protein.

Vasodilatation and inhibition of mediator release represent two distinct mechanisms for prostaglandin modulation of acute mast cell‐dependent inflammation

1. Intravital microscopy of the hamster cheek pouch was used to examine the influence of vasodilator prostanoids (prostaglandin E2 (PGE2), PGI2), forskolin, and nitroprusside on the microvascular changes during acute inflammation induced by antigen or histamine. The results extend our previous finding that PGE2 modulates allergic inflammation and histamine release in the cheek pouch model. 2. The microvascular actions of arachidonic acid and different cyclo-oxygenase products (PGE2, PGD2, PGI2, PGF2 alpha, and the thromboxane A2 (TXA2)-analogue U-44069) were first compared with respect to their effects on arteriolar tone. Of the prostaglandins, only PGE2 and PGI2 were potent vasodilators and markedly increased local blood flow. Nitroprusside and forskolin also caused vasodilatation and increased blood flow, but were somewhat less potent than PGE2 and PGI2. 3. Topically applied PGE2 and PGI2 in vasodilator concentrations suppressed the antigen-induced plasma leakage. On the other hand, although the antigen response was predominantly mediated by histamine, both prostaglandins enhanced the plasma leakage evoked by exogenous histamine. 4. In contrast, the vasodilator nitroprusside, in a dose causing an increase in blood flow equal to that of PGE2 and PGI2, potentiated both the histamine-induced plasma leakage, as well as the plasma and leukocyte extravasation after antigen challenge, indicating that the anti-inflammatory actions of the prostaglandins were unrelated to their vasodilator properties per se. 5. Because forskolin, a specific activator of adenylate cyclase, mimicked the actions of PGE2 and PGI2, i.e. inhibition of the antigen-induced plasma extravasation and enhancement of the histamine response, it is possible that the observed antiallergic effects of the prostaglandins were related to accumulation of intracellular adenosine 3': 5'-cyclic monophosphate (cyclic AMP). 6. Taken together, there appears to be a competition between pro- and anti-inflammatory effects of PGE2 and PGI2 in reactions involving release of endogeneous inflammatory mediators in vivo, i.e. enhancement of inflammatory mediator target action on one hand ('two mediator synergism'), and suppression of mediator release on the other. Moreover, the observations indicate that vasodilatation and inhibition of mediator release are two distinct actions of PGE2 and PGI2.

Photoendocrine transduction in cultured chick pineal cells. II. Effects of forskolin, 8-bromocyclic AMP, and 8-bromocyclic GMP on the melatonin rhythm

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin release, and the effects of light and darkness upon it. Here, we describe the effects of forskolin (FSK), a specific activator of adenylate cyclase, 8-bromocyclic AMP (8BrcA), an analogue of cyclic AMP, and 8-bromocyclic GMP (8BrcG), an analogue of cyclic GMP, on melatonin output by these cells. FSK stimulated melatonin output; it was potent and effective. 8BrcA, but not 8BrcG, also markedly stimulated melatonin output. These results support a role for cAMP (but not cGMP) in the regulation of melatonin production. Four hour pulses of white light or darkness, in otherwise constant red light, cause, in addition to acute effects, phase-dependent phase shifts of the melatonin rhythm in subsequent cycles. Such phase shifts indicate an effect on (proximal to) the pacemaker generating the rhythm. Four or 8 hour pulses of FSK, 8BrcA, or 8BrcG, however, did not appreciably alter the phase of subsequent melatonin cycles. Neither did they interfere with phase shifts induced by light pulses. These results fail to support a prominent role for cAMP or cGMP levels in regulating the pacemaker; nor do these cyclic nucleotides appear to mediate the prominent effects of light and dark on the pacemaker. Thus, cyclic AMP regulation of melatonin appears to occur distal to the pacemaker.

Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists

Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10−4 M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/ min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10−4 M). vasoactive intestinal polypeptide (10−6 M) and isoproterenol (10−5 M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopaminesensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction. These findings suggest the presence of discrete populations of growth cones in developing rat forebrain that contain either dopamine, vasoactive intestinal polypeptide or β-adrenergic receptors coupled to adenylate cyclase.

Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membranes of freshwater-adapted trout by measuring the conversion of [α- 32P]ATP into [α- 32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20°. The K m for ATP was 0.5 m M in the presence or absence of the stimulators. The presence of 10 −5 M guanosine-5′-triphosphate and 4 × 10 −3 M MgCl 2 (2.41 × 10 −3 M free Mg 2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca 2+ was inhibitory. IC 50 for CaCl 2 was 5 × 10 −4 M (10 −7 M free Ca 2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400–450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE 1 10 −5 M (50% increase over basal activity) or by glucagon 5.7 × 10 −10 M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10 −2 M.

Effects of purines and forskolin on haemolysis.

Effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine on the antihaemolytic action of procaine, and the effect of ATP on the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine were investigated in rat erythrocytes. The effects of adenosine and its analogues D-N6-phenylisopropyladenosine (D-PIA), L-N6-phenylisopropyladenosine (L-PIA), N6-cyclohexyladenosine (CHA), 2-chloroadenosine, and N6-methyladenosine, as well as the effects of cyclic-AMP (cAMP), dibutyryl cAMP, and forskolin on haemolysis were also investigated in rat erythrocytes. ATP, ADP, and AMP, but not adenosine, antagonized in a concentration-dependent manner the antihaemolytic action of procaine, and ATP was ineffective against the antihaemolytic actions of lidocaine, dibucaine, tetracaine, pentobarbitone, and chlorpromazine. Adenosine and its analogues, but not N6-methyladenosine, protected erythrocytes against hypotonic haemolysis in a concentration-dependent manner. The order of potencies was: D-PIA greater than CHA greater than L-PIA greater than adenosine greater than 2-chloroadenosine. The effect of adenosine on haemolysis was not prevented by theophylline, 8-phenyltheophylline, or 8-sulfophenyltheophylline. Dibutyryl cAMP, a stable analogue of cAMP, and forskolin, a specific activator of adenylate cyclase, mimicked the antihaemolytic action of adenosine. cAMP was less efficient than adenosine. Adenine nucleotides antagonized the antihaemolytic action of procaine, probably due to calcium-chelating properties. In contrast, adenine nucleosides have antihaemolytic properties. The possibility that the antihaemolytic effects of these substances relate to cAMP and/or to lipophilicity is discussed.

Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses.

There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH.

Prolongation of calcium action potentials by gamma‐aminobutyric acid in primary sensory neurones of lamprey.

Intracellular recordings from primary mechanosensory neurones (dorsal cells) in the lamprey spinal cord were used to test the membrane effects of a variety of putative neuromodulatory agents. gamma-Aminobutyric acid (GABA) produced a dose-dependent increase in the duration of mixed Na-Ca or pure Ca action potentials in these cells. L-Glutamate and glycine produced minimal broadening of Ca action potentials. Acetylcholine, noradrenaline, serotonin, met-enkephalin, D-glutamate and dopamine had no effect. The pharmacology of GABA's action appeared to be complex. While the GABAA receptor antagonists, bicuculline, picrotoxin and curare, did not block GABA's effect, both the GABAA receptor agonist, muscimol, and the GABAB-receptor agonist, baclofen, occasionally broadened Ca action potentials in these cells. GABA had no effect on the resting potential, passive current-voltage (I-V) characteristics and pure Na action potential of dorsal cells, ruling out an action on passive membrane channels, transmitter-activated channels, or on those voltage-dependent channels activated during the Na action potential. Thus, GABA affected dorsal cells only when a significant Ca current was evident. GABA appeared not to increase the conductance of the Ca channels since its action was accompanied by an increase in input resistance, suggesting an inhibition of Ca-dependent conductance that normally acts to repolarize the membrane during a Ca action potential. An inhibitory effect of GABA on a Ca-dependent Cl conductance was ruled out in experiments where the Cl gradient was altered by removal of extracellular Cl without affecting GABA-induced Ca action potential prolongation. Dorsal cells have a prominent Ca-dependent K conductance (gK(Ca], and it is this conductance that GABA may inhibit. Consistent with this was the observation that the hyperpolarizing after-potential that follows Ca action potentials in dorsal cells, which reflects gK(Ca) in these cells and whose duration is normally increased when the Ca action potential duration increases, was not prolonged when the Ca action potential was broadened by GABA. Further, the failure of GABA to prolong Ba action potentials was consistent with this proposed mechanism of action, since Ba apparently does not activate gK(Ca) in these cells. Forskolin, a specific adenylate cyclase activator, caused broadening of Ca action potentials in lamprey dorsal cells comparable in magnitude to that of GABA. Thus, an increase in intracellular cyclic AMP is a candidate for the intracellular mediator of GABA's effect on these cells.