Amount Of Cyclic AMP Research Articles

Parathyroid hormone increases the sensitivity of inositol trisphosphate receptors by a mechanism that is independent of cyclic AMP.

1 In fura 2-loaded HEK-293 cells stably expressing human type 1 parathyroid hormone (PTH) receptors, PTH potentiated the Ca(2+) mobilization evoked by carbachol by >4 fold without itself increasing the intracellular [Ca(2+)]. 2 PTH potentiated the Ca(2+) release evoked by a cell-permeant analogue of inositol 1,4,5-trisphosphate (InsP(3)BM). 3 Prolonged incubation with InsP(3)BM emptied the Ca(2+) stores as effectively as PTH in combination with a maximal concentration of carbachol, indicating that PTH did not increase the size of the InsP(3)-sensitive Ca(2+) pool. 4 Responses to PTH were unaffected by disruption of the cytoskeleton. 5 The EC(50) for carbachol-evoked Ca(2+) release and InsP(3) formation were indistinguishable (approximately 40 microM), consistent with even the highest concentrations of carbachol generating insufficient InsP(3) to release the entire InsP(3)-sensitive Ca(2+) pool. 6 Inhibition of cyclic AMP-dependent protein kinase A (PKA), using H89 or CMIQ, did not affect potentiation of carbachol-evoked Ca(2+) signals by PTH. 7 SQ22536 or DDA, inhibitors of adenylyl cyclase, inhibited PTH-evoked cyclic AMP formation and IBMX, an inhibitor of cyclic nucleotide phosphodiesterase, increased the amount of cyclic AMP detected after stimulation by PTH. None of these drugs affected the potentiation of Ca(2+) signals by maximal or submaximal concentrations of PTH. 8 We conclude that PTH potentiates the Ca(2+) release evoked by receptors that stimulate InsP(3) formation by sensitizing InsP(3) receptors through a cyclic AMP-independent mechanism.

Cyclic AMP, a possible regulator of glycolysis in the ectomycorrhizal fungus Amanita muscaria.

The amounts of cyclic AMP (cAMP), fructose-2,6-bisphosphate (F26BP), trehalose and glycogen were determined in cell suspension cultures of the ectomycorrhiza-forming fungus Amanita muscaria (L. ex Fr.) Hooker. For the assay of cAMF a protocol was developed that enabled the detection of as little as 50 fmol of this secondary messenger by an enzyme-linked immuno assay (EIA). Values varied from < 1 and up to 5 pmol cAMP mg1 d. wt according to the age of the fungal culture. Typically, a transient increase in cAMP occurred after c. 4 d of culture of the fungus on glucose-containing medium. This increase (up to 100%) was followed by the start of the logarithmic growth phase, and by a more persistent increase in F26BP. In parallel, glucose in the medium started to decrease, whilst the amounts of fungal carbohydrates, especially the disaccharide trehalose, increased, From these data we assume that a high initial rate of glucose uptake caused an increase in the fungal pools of storage carbohydrates and, via activation of an adenylate cyclase, of cAMP. According to data reported for yeast cells this should enhance the formation of F26BP by phosphorylation of relevant enzymes. In animal and yeast cells an increase in the concentration of F26BP stimulates glycolysis by activation of the ATP-dependent phosphofructokinase (PFK). A. muscaria also possesses an F26BP activated PFK and, under conditions of symbiosis, host-derived carbohydrates are supplied mainly in the form of glucose. The implications of these findings to the regulation of carbohydrate metabolism of symbiotic plant root/fungus structures (ectomycorrhiza) are discussed.

Fuc-GM1 ganglioside mimics the receptor function of GM1 for cholera toxin.

The ability of Fuc-GM1 ganglioside to mimic the receptor function of GM1 for cholera toxin (CT) has been investigated. For this purpose, rat glioma C6 cultured cells were enriched with Fuc-GM1 and the responsiveness to CT was compared with that of cells enriched with GM1 ganglioside. Fuc-GM1 was taken up by cells as rapidly and to the same extent as GM1. When comparable amounts of ganglioside were associated, the cells enriched with Fuc-GM1 bound the same amount of 125I-CT as did cells enriched with GM1. Under conditions in which GM1- and Fuc-GM1-enriched cells bound comparable amounts of CT, the Fuc-GM1-treated cells accumulated virtually the same amount of cyclic AMP as did GM1-treated cells, and activation of adenylate cyclase was also similar. The lag time preceding the CT-induced cAMP accumulation was the same in Fuc-GM1- and GM1-enriched cells. High-sensitivity isothermal titration calorimetry (ITC) experiments showed that the association constants of CT with Fuc-GM1 or GM1 ganglioside were comparable (4 x 10(7) M-1 and 1.9 x 10(7) M-1, respectively, at 25 degrees C). Also, the association constants of the B-subunit pentamer with Fuc-GM1 or GM1 ganglioside were comparable (about 3 x 10(7) M-1 and 7 x 10(7) M-1, respectively, at 25 degrees C).

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Bordetella pertussis Adenylate Cyclase Activity in Nasopharyngeal Aspirates for Rapid Diagnosis of Whooping Cough in Relation to Culture and Serology

Adenylate cyclase activity, measured in 201 nasopharyngeal aspirates from patients presenting with own or parental suspicion of whooping cough, was compared to diagnosis made by culture and by serology in the culture negative cases. The median amount of cyclic AMP in samples from culture negative patients (n = 145) was 0.60 pmoles which differed significantly (p less than 0.001) from the median value 3.28 in samples from culture positive patients (n = 56). The median value 0.70 pmoles of cyclic AMP in samples from culture negative patients who were positive by serology (n = 54) did not differ significantly from the value of 0.57 pmoles in samples from serology negative patients (n = 91). With a limit for positive cyclic AMP set at 2 pmoles, 45 samples were positive. The sensitivity of the assay was 66% (37/56) in culture positive patients while the specificity was 93% (85/91) in the serology negative patients. The positive predictive value for the c-AMP test was 82% (37/45) in relation to culture and 87% (39/45) in relation to culture and/or serology. The results confirmed that measurement of adenylate cyclase activity in nasopharyngeal aspirates by an 1-h incubation method can serve as an early and rapid diagnostic method of pertussis infection. The low sensitivity of the c-AMP assay in samples from serology positive but culture negative patients indicates however, that this assay will have to be supplemented by serology for a high diagnostic sensitivity in all cases of pertussis.

Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters

The liver tumor promoters phenobarbital (PB) (20–500 μg/ml) and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (1–10 μg/ml) inhibited intercellular communication between primary cultured B6C3F1 mouse hepatocytes after 8 hr of treatment. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabeled donor hepatocytes to recipient hepatocytes. The addition of either dibutyryl cyclic AMP (N6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) (0.001–0.1 mm) or caffeine (0.01–1 mm) decreased or completely abolished the inhibitory effects of PB and DDT on intercellular communication. Cyclic AMP (adenosine 3′:5′-cyclic monophosphate; cAMP) in primary cultured mouse hepatocytes was measured by radioimmunoassay. Cyclic AMP in nontreated, freshly plated cultures declined from 4.2 ± 0.7 pmol/mg protein after 1 hr in culture to 2.4 ± 0.5 pmol/mg protein after 8 hr in culture. Phenobarbital at 250 and 500 μg/ml significantly decreased cyclic AMP below control values after 1 hr of treatment. However, no difference in the amount of cyclic AMP was detected between control and PB-treated cultures after 2, 4, and 8 hr in culture or with lower PB concentrations. DDT at 10 μg/ml decreased cAMP levels in the hepatocytes after 1, 2, 4, and 8 hr of treatment. No effects were seen after 8 hr of treatment or with lower DDT concentrations. DDT (10 μg/ml) also decreased cAMP levels in 24-hr-old cultures while PB (500 μg/ml) had no effect. Addition of dibutyryl cAMP (0.1 mm) or caffeine (1.0 mm) to freshly plated cultures elevated cAMP levels 50-fold and twofold, respectively. These data suggest that the inhibition of mouse hepatocyte intercellular communication by PB and DDT at the highest concentrations tested may be mediated by transient decreases in intercellular cAMP levels.

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells.

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.

Nitroglycerine- and isoprenaline-induced vasodilatation: assessment from the actions of cyclic nucleotides.

To investigate the vasodilator actions of nitroglycerine and isoprenaline, the effects of these agents, dibutyryl cyclic AMP (db cyclic AMP) and 8-bromo cyclic GMP (8-Br cyclic GMP) on intact muscle tissue, and cyclic AMP and cyclic GMP on skinned muscle of the rabbit mesenteric artery were investigated. In porcine coronary artery, nitroglycerine (greater than 0.1 microM) increased the production of cyclic GMP with no change in the amount of cyclic AMP, while isoprenaline (greater than 0.1 microM) significantly increased the production of cyclic AMP with no change in the amount of cyclic GMP. In the rabbit mesenteric artery, nitroglycerine or isoprenaline inhibited the tonic component of the 39 mM [K]o-induced contraction to a greater extent than the phasic component. Nitroglycerine and 8-Br cyclic GMP showed a stronger inhibitory action on the K-induced contraction than did isoprenaline and db cyclic AMP. The sources of Ca utilized for the generation of contraction by noradrenaline and caffeine were estimated to be the same as those determined from the amplitudes of contractions evoked in Ca-free solution by various concentrations of noradrenaline or caffeine. In intact muscle tissues, the effects of nitroglycerine or 8-Br cyclic GMP on the amount of Ca stored in cells were estimated from the caffeine-induced contraction in Ca-free solution. Both agents inhibited the contractions due to a reduction in the amount of Ca in the cells. When the effects of isoprenaline or db cyclic AMP were observed, both agents inhibited the caffeine-induced contraction but the accumulation of Ca into cells was greater than the control. In saponin skinned muscles, the pCa-tension relationship in the presence of cyclic AMP and cyclic AMP-dependent protein kinase (cyclic AMP-PK) shifted to the right and to a lower level in comparison with the control. Applications of cyclic GMP with cyclic GMP-dependent protein kinase (cyclic GMP PK) also inhibited the contraction induced by low concentrations of Ca. In skinned muscles, cyclic AMP exhibited dual actions on Ca store sites, i.e. in the presence of high concentrations of Ca or prolonged superfusion of Ca, cyclic AMP reduced the amount of Ca due to activation of the Ca-induced Ca release mechanism by excess accumulation of Ca. On the other hand, cyclic GMP consistently inhibited the amplitude of the caffeine-induced contraction due to a reduction in the amount of Ca in the store sites. 8 These results indicate that nitroglycerine and isoprenaline increase the amount of cyclic GMP and cyclic AMP, respectively. The main effect of cyclic GMP is activation of Ca extrusion, thus reducing the amount of Ca stored in the cell, while the main effect of cyclic AMP is to increase the amount of Ca stored in the cell. Both cyclic AMP with cyclic AMP-PK and cyclic GMP with cyclic GMP-PK inhibit the phosphorylation of myosin. Consequently both cyclic nucleotides reduce the free Ca in the myoplasm and promote relaxation, but by different mechanisms.

Beta-adrenergic stimulation of prostaglandin production by human amnion tissue

Arachidonic acid is released from specific glycerophospholipids in human amnion and is used to synthesize prostaglandins that are involved in parturition. In an investigation of the regulation of prostaglandin production in amnion, the effects of isoproterenol on discs of amnion tissue maintained in vitro were examined. Isoproterenol caused a large but transitory increase in the amount of cyclic AMP in amnion discs and this was accompanied by a sustained stimulation of the release of arachidonic acid (but not palmitic acid or stearic acid) and prostaglandin E 2. The dependencies of cyclic AMP accumulation, arachidonic acid mobilization and prostaglandin E 2 release on the concentration of isoproterenol were similar, each response was maximal at 10 −6 M isoproterenol and was inhibited by propranolol. Dibutyryl cyclic AMP stimulated the release of prostaglandin E 2 from amnion discs. Although prostaglandin E 2, when added to amnion discs caused an accumulation of cyclic AMP, it did not appear to mediate isoproterenol-induced accumulation of cyclic AMP since the latter effect was insensitive to indomethacin in concentrations at which prostaglandin production was inhibited greatly. These data support the proposition that catecholamines, found in increasing amounts in amniotic fluid during late gestation, my be regulators of prostaglandin production by the amnion.

Forskolin, cyclic nucleotides and positive inotropism in isolated papillary muscles of the rabbit.

The effects of forskolin, isoprenaline, sodium nitroprusside and the frequency of stimulation were examined on cyclic nucleotide levels and tension responses in rabbit isolated right ventricular papillary muscles. Increasing the frequency of stimulation from 0.01 Hz to 1.6 Hz induced positive inotropic responses that were not obviously related to alterations in the level of either cyclic AMP or cyclic GMP. Isoprenaline induced rapid, concentration-related positive inotropic responses that were associated with increases in the levels of both cyclic AMP and cyclic GMP. There existed good correlations between the increases in tension and the concentrations of both cyclic nucleotides measured in the tissues. Forskolin induced concentration-related positive inotropic responses that were slow to develop. These responses were accompanied by concentration-related increases in the levels of cyclic AMP but not cyclic GMP. The tension responses correlated well with the levels of cyclic AMP measured. The cyclic AMP levels produced by forskolin were some 8 fold higher than those induced by isoprenaline for similar increases in tension. Sodium nitroprusside was without inotropic effect either positive or negative; it nevertheless elevated cyclic GMP levels whilst slightly reducing cyclic AMP levels. These data show that the ratio of cyclic AMP to cyclic GMP does not correlate well with changes in mammalian cardiac contractility. The data further suggest that whilst the intracellular concentration of cyclic AMP in rabbit ventricular myocardium may be an important determinant of positive inotropism, the relationship between the two parameters is more complex than simple proportionality between the tension generated and the amount of cyclic AMP measured within the cells.

Cyclic AMP as a mitotic signal for epidermal keratinocytes, but not for dermal fibroblasts.

The function of cyclic AMP in the growth of epidermal and dermal cells was investigated. Cholera toxin was used to increase the amount of intracellular cyclic AMP. This toxin had a stimulatory effect on human epidermal cells only when growth was limited; i.e., when a small number of cells was plated for colony formation, when cells from frozen stock were cultured, and when cells were in the initial phase of primary culture. Cholera toxin, however, had no significant effect on the growth rate during exponential growth or on the saturation density during the stationary phase.The stimulatory effect of the toxin was specific to epidermal keratinocytes. In other types of cells, the effect varied : with human dermal fibroblasts there was no ffeect, or some inhibition.A membrane receptor for cholera toxin, GM 1 ganglioside, was isolated from human epidermal cells. Cyclic AMP was induced markedly by cholera toxin in human epidermal keratinocytes, which suggests that an increase in the amount of cyclic AMP may act as a mitotic signal in these cells. Cholera toxin also induced cyclic AMP in human dermal fibroblasts irrespective of the growth response, an indication that in dermal fibroblasts the content of cyclic AMP is not necessarily related to the proliferation of the cells.

Effects of uridine triphosphate on contractility, cyclic nucleotide levels and membrane potential in the isolated frog ventricle

A study has been made of the effects of uridine 5'-triphosphate (UTP) on the isolated frog ventricle. Preparations were superfused with solutions containing different concentrations of UTP, and changes in contractility, cyclic nucleotide levels and membrane potential were measured. UTP produced a long lasting increase in isometric twitch tension, which was unaffected by adrenergic receptor antagonists (propranolol and phentolamine). The levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of the ventricle to 10(-4) M UTP. The increase in the force of contraction was found to be accompanied by a rise in intracellular cyclic AMP. Cyclic GMP levels were seen to fall initially, but then to increase later, as both twitch and cyclic AMP started to decline. UTP also produced marked changes in the shape of the action potential; its duration and positive overshoot were both increased. The effects of UTP on twitch tension, cyclic nucleotide levels and action potential parameters were all dose-related. The change in contractility was found to be linearly related to (a) the ratio of the amount of cyclic AMP: cyclic GMP present in the fibres, and (b) to the increase in duration of the action potential. These results suggest that cyclic AMP, cyclic GMP and the availability of Ca2+ may all be involved in mediating the response to UTP.

Cyclic 3′,5′-adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat

Intracellular content of cyclic 3′,5′-adenosine monophosphate was determined in several strains of Trypanosoma brucei brucei during their growth in rats. In non-relapsing infections with the cloned monomorphic strain 110M and with the cloned pleomorphic strain YTatl, the amount of cyclic AMP per 10 9 trypanosomes increased from 30 to over 90 pmol as the parasitemia increased from patency to over 10 9 organisms per ml. This increase was not observed during non-relapsing infections with strain 427. During infections with strain YTatl in both immunocompetent and lethally X-irradiated rats, cyclic AMP content of the parasite increased from 20-20 pmol per 10 9 cells early in logarithmic growth to 65–70 pmol per 10 9 cells at peak parasitemia, then decreased as the transition to intermediate and short stumpy forms commenced. At crisis, basal levels were reestablished when long slender forms were the lowest percentage of the total population and intermediate and short stumpy forms predominated, suggesting a correlation between morphologic type and level of cyclic AMP per cell during fluctuations in parasitemia. Increases in intracellular cyclic AMP were measured during in vitro incubation of the parasite in medium containing potential effectors of the trypanosome cyclic AMP system. Sodium fluoride, adenosine and methyl xanthines stimulated increases in cyclic AMP content while isoproterenol, prostaglandin E 1, serotonin, histamine and several trypanocidal drugs were ineffective. The results are discussed in terms of the possible regulatory role of cyclic AMP in differentiation of trypanosomes.

Post receptor activation of lipolysis in starvation, diabetes mellitus and hyperthyroidism.

Activation of lipolysis by cyclic AMP in conditions with accelerated lipid mobilization was examined in subcutaneous adipose tissue incubated in vitro. In (a) 16 obese patients before and during therapeutic starvation, (b) 18 diabetics before and after antidiabetic treatment and (c) 11 hyperthyroid patients before and after anti-thyroid treatment, a positive correlation was found between stimulation of basal cyclic AMP accumulation and stimulation of basal glycerol release using either isopropyl noradrenaline or noradrenaline (r = 0.6-0.9). During antidiabetic treatment stimulation of lipolysis increased in relation to that of cyclic AMP accumulation (F = 10.1, p less than 0.01), whereas during antithyroid therapy there was a decrease (F = 95.2, p less than 0.01). Starvation did not alter the relationship between lipolysis and cyclic AMP in hypogastric adipose tissue whereas in femoral tissue stimulation of lipolysis decreased in relation to that of cyclic AMP accumulation (F = 9.6, p less than 0.01). It is concluded that the amount of cyclic AMP needed to promote lipolysis is increased during starvation and in diabetes mellitus but is decreased in hyperthyroidism. From the studies during starvation it appears that regional differences in the post-receptor activation of lipolysis exist in human adipose tissue.

Culture sensitization and inhibition of luteinizing hormone responsive production of cyclic AMP in luteal cells by luteinizing hormone, prostaglandin F2 alpha and [D-Trp6]-luteinizing hormone releasing hormone.

Many cells are able to regulate their sensitivity to hormones. In order to investigate the mechanism(s) by which rat luteal cells regulate their sensitivity to LH, we have developed and characterized a cell culture model. Cultures of dispersed rat luteal cells were exposed to graded doses of bovine LH, an analogue of LH releasing hormone (LH-RH) ([D-Trp6]-LH-RH), and prostaglandin F2 alpha (PGF2 alpha) for 3 h. The media containing these hormones were then replaced with fresh hormone-free medium and the cells cultured for 24 h. In order to test the sensitivity of these cultures after 24 h, the medium was discarded and replaced by medium alone, or medium containing a standard dose of bovine LH for 1 h. The amount of cyclic AMP accumulated during this hour was used as an index of the sensitivity of the cells to LH. Control cultures became 'supersensitive' to LH with augmented production of cyclic AMP during culture but LH-receptor binding activity was not increased. During the first 2 h of culture, LH (100 ng/ml) increased accumulation of cyclic AMP by fourfold, but after 5 h of culture, stimulation of cyclic AMP accumulation by the same dose of LH was increased 32-fold, 299-fold at 13 h and 359-fold at 21 h of culture. The increase in LH-responsive accumulation of cyclic AMP with cultured was severely impaired by early exposure of cells to LH, LH-RH analogue or PGF2 alpha during the first 2 h of culture. Also, both LH-RH analogue and PGF2 alpha acutely inhibited LH-stimulated accumulation of cyclic AMP. Inhibition of culture-induced sensitization of LH responsiveness was not altered by the addition of 3-isobutyl-1-methylxanthine. Scatchard analysis of LH binding sites indicated that pretreatment of luteal cells with LH (or human chorionic gonadotrophin at an equivalent dose) reduced the number of free LH receptors when measured after 24 h of culture, but total receptor binding activity was not changed. However, a similar effect was not seen with cells treated with PGF2 alpha or LH-RH analogue. It is suggested that 'culture-induced' supersensitivity may represent either recovery of pre-isolation sensitivity or result from the loss of an endogenous factor(s) which retards the coupling of the LH receptor and adenylate cyclase. Although PGF2 alpha and LH-RH analogue have been shown to directly prevent the occupied LH receptor from activation of adenylate cyclase, the present observations have indicated that this inhibitory process was continued even when these agents were removed from the culture medium.

Adrenocortical responses to adrenocorticotrophin in the rat.

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.

Subpopulations of T lymphocytes from human blood differing in density and stage of maturation.

Previous investigations have shown that peripheral lymphocytes with a low specific gravity are much more active than are heavy lymphocytes in the proliferative response towards mitogens, antigens and allogeneic lymphocytes. We have now studied the biological significance of this difference in human peripheral T lymphocytes with two independent parameters generally regarded as maturation criteria. Heavy human T lymphocytes contain about one-third of the amount of cyclic AMP found in light T cells, but the increase in cyclic AMP produced by isoproterenol or prostaglandin E1 is much higher in the heavy T cells. The lactate dehydrogenase isoenzyme pattern of heavy human T lymphocytes is intermediate between that of human thymocytes and light T lymphocytes, the latter showing the most mature pattern. A high correlation was found between these two parameters, the cell density and the reactivity towards phytohemagglutinin. We conclude that human peripheral T lymphocytes are heterogeneous in maturation level: heavy, nonreactive T lymphocytes are less mature than light T lymphocytes.

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.

Serum thymic factor in subacute sclerosing panencephalitis patients

The activity of thymus-dependent serum factor (SF) was measured in 11 patients with subacute sclerosing panencephalitis (SSPE). A significantly decreased activity was found in 6 out of 11 patients, whereas the level of cyclic AMP in lymphocytes of the patients, a parameter for the maturation of peripheral blood lymphocytes, showed low values in 9 out of 11 patients. These findings were compared with the clinical stage of the disease and with some lymphocyte functions in vitro. No correlation was found between the SF levels and the amount of cyclic AMP in the lymphocytes or between these two parameters and the lymphocyte functions in vitro. It is concluded that a thymic humoral dysfunction is unlikely to play a role in the pathogenesis of SSPE; it appears that such a dysfunction is rather a secondary event in this disease.

EFFECT OF LUTEINIZING HORMONE ON THE CONCENTRATION OF CYCLIC GMP IN RABBIT OVARIES

Laboratoire d'Endocrinologie, Hôpital Notre-Dame et Département de Medicine, Université de Montréal, Montréal, Canada (Received 28 June 1978) Cyclic AMP has been implicated as an intermediate in some of the actions of luteinizing hormone (LH) on ovarian tissues, such as stimulation of steroidogenesis (LeMaire &amp; Marsh, 1975). Both in vitro (Marsh, Butcher, Savard &amp; Sutherland, 1966) and in vivo (Armstrong, Dorrington &amp; Robinson, 1976), stimulation with LH results in a rapid increase in the amount of cyclic AMP in ovarian tissues, which precedes the LH-induced increase in steroidogenesis. Recently, studies on rat ovaries (Grinwich, Ham, Hichens &amp; Behrman, 1976; Ratner, 1976; Ratner &amp; Sanborn, 1976) have indicated that the ovarian tissue content of cyclic GMP may also be regulated by LH, but in a direction opposite to that of cyclic AMP. In the rabbit, Goff &amp; Major (1975) have shown that administration of human chorionic gonadotrophin (HCG) causes a biphasic increase