Content and metabolism of cyclic AMP at different stages of growth of hepatoma 22a

The intracellular content of cyclic 3,5-adenosine monophosphate (AMP) and activity of adenylate cyclase and cyclic AMP phosphodiesterase were determined in the lag period and the exponential and stationary phases of growth of mouse hepatoma 22a. In the stage of transition of the tumor cells from the lag period to the exponential phase of growth, the intracellular cyclic AMP concentration was found to be reduced by half as the result of sudden activation of phosphodiesterase. Later the cyclic AMP content fell more slowly until the cells entered the stationary phase of growth. Since adenylate cyclase activity remained unchanged during growth of the hepatoma 22a, this suggests that an increase in phosphodiesterase activity is the signal for escape of the tumor cells from the resting period and their entry into the mitotic cycle.Laboratory of Biochemical Pharmacology, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny Vol. 84, No. 10, pp. 472–474, October, 1977.     

Vasoactive hormones and cAMP affect pericyte contraction and stress fibresin vitro

Summary Pericytes are contractile cells of the microvascular wall that may influence capillary haemodynamics and permeability. We examined the contractile responses of cultured pericytes to selected vasoactive agents and cAMP agonists. Morphological and biochemical changes associated with these responses were also studied. Pericytes seeded onto silicone rubber contracted when stimulated with histamine or serotonin, relaxed in response to the beta-adrenergic agonist isoproterenol and did not respond to epinephrine. Since hormonal-induced relaxation of vascular smooth muscle involves cAMP, we investigated the ability of cAMP, to modulate pericyte contraction. Dibutyryl cAMP and forskolin (an adenylate cyclase activator) both induced pericyte relaxation and elevated intracellular cAMP levels. Isoproterenol increased cAMP levels but epinephrine had no effect. However, when epiniphrine and isoproterenol were co-incubated with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX), cAMP was increased to levels above those elicited by these agonists alone. Serotonin and histamine in the presence of IBMX did not affect cAMP levels. These results suggest that certain vasoactive agents may relax pericytes by cAMP-dependent processes. We have shown previously that stress fibres are also involved in pericyte contraction. Hence, changes in the staining patterns of stress fibres in response to these selected agonists were studied. Histamine, serotonin and epinephrine had no apparent effect on stress fibre staining. Dibutyryl cAMP, forskolin, and isoproterenol, which relax pericytes and increase cAMP, disassembled fibres. In summary, the results demonstrate that the contractile activity of cultured pericytesin vitro can be regulated by vasoactive agonists and that changes in cAMP and stress fibres may mediate the regulation.Abbreviations cAMP cyclic adenosine 3,5-monophosphate - dBcAMP dibutyryl cyclic adenosine 35-monophosphate - DiL-Ac-LDL 1,1-dioctadecyl 1-3,3,3,3 tetramethylindo-carbocyanine perchlorate - DME Dulbecco's Modified Eagles Medium - FCS foetal calf serum - HBSS Hanks balanced salt solution - IBMX 3-isobutyl-1-methylxanthine - PBS phosphate buffered saline - R020-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone - TCA trichloroacetic acid     

Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.     

Cyclic nucleotides and vasoconstrictor function: physiological and pathophysiological considerations

The role of cyclic nucleotides as modulators of vascular smooth muscle tone has been widely studied. Yet the cellular mechanisms whereby cAMP and cGMP promote vascular relaxation remain highly controversial. The purpose of this review is to summarize a large and expanding literature with particular emphasis on the cellular mechanisms of cAMP- and cGMP-induced relaxation of vascular smooth muscle. The review addresses the following topics: regulation of vascular tone, mechanisms of cAMP- and cGMP-mediated vascular smooth muscle relaxation, modulation of [Ca²⁺]_i by cAMP and cGMP, effects of cAMP and cGMP on the Ca²⁺ sensitivity of the contractile apparatus, and pathophysiology of the resistance vasculature in chronic portal hypertension with particular emphasis on cAMP and cGMP dependent pathways.     

Effect of cyclic AMP,db-cyclic AMP and phosphodiesterase inhibitors on histamine inhibition of the contractile response of the mouse vas deferens

Histamine inhibited the contractile response of the mouse vas deferens to various frequencies of stimulation, and the inhibition was inversely related to the frequency of stimulation. This effect of histamine was mimicked by cyclic AMP, db-cyclic AMP and various phosphodiesterase inhibitors (IBMX, aminophylline and theophylline). Histamine-produced inhibition, but not that produced by the other compounds, was blocked by cimetidine. The low concentrations (10–100 M) of various phosphodiesterase inhibitors caused inhibition but failed to potentiate the inhibitory response to histamine. The basal cyclic AMP levels of the tissues were unaffected at these concentrations of aminophylline. At higher concentrations (1 and 5 mM), however, aminophylline significantly elevated the basal cyclic AMP levels of the tissues and markedly inhibited the contractile response to various frequencies of stimulation but still failed to enhance or potentiate the inhibitory response to histamine. In fact, the inhibitory response to histamine at these concentrations of aminophylline was reduced. Based on these and earlier [3] findings, it is concluded that, although histamine increases the accumulation of cyclic AMP in the mouse vas deerens and although its inhibitory effect on the preparation can be mimicked by both the cyclic nucleotides and phosphodiesterase inhibitors, the involvement of cyclic AMP in the mediation of its inhibitory response is still unresolved.     

Regulatory role of dopa and components of the cyclic AMP system

Injection of nonradioactive dopa (1 mg/mouse) before injection of [³H]dopa (20 Ci/mouse) into albino mice with Harding-Passy melanoma leads to increased accumulation of tritium in the tumor tissue. Radioactivity of the melanoma in this experimental group was twice as high as the radioactivity of the tumor tissue in animals receiving injections of [³H]dopa alone. Investigation of adenylate cyclase and phosphodiesterase activity and the cyclic AMP level in the melanoma of the mice 2 h after injection of dopa (1 mg/mouse) revealed accumulation of cyclic AMP and increased phosphodiesterase activity; adenylate cyclase activity was depressed. It is suggested that dopa exerts its effect not only as a precursor of melanin, but also through the cyclic AMP system, affecting the activity of enzymes of melanogenesis.Deceased.Department of Biochemistry, S. M. Kirov Military Medical Academy, Leningrad. V. G. Khlopin Radium Institute, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Klimov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 425–426, April, 1978.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that -SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to -SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

Dual regulation of M current in gastric smooth muscle cells: β-adrenergic-muscarinic antagonism

The effects of the -adrenergic agent isoproterenol on membrane currents were studied in freshly dissociated gastric smooth muscle cells of Bufo marinus. Voltage-clamp experiments were carried out with patch pipettes in the tight-seal, whole-cell recording mode or with conventional microelectrodes. Isoproterenol induced a current identified as M current by the following criteria: the induced current is outward and carried by K⁺ ions, is suppressed by muscarine or acetylcholine, remains steadily activated, turns off with hyperpolarization, and exhibits slow relaxations in response to voltage jumps. In contrast to endogenous M current, isoproterenol-induced M current usually exhibited slower relaxations on hyperpolarizing voltage commands and displayed a steady-state conductance/voltage relationship that was shifted in the negative direction along the voltage axis. M current was also induced by either forskolin or phosphodiesterase-resistant cAMP analogs. In all cases, muscarinic agonists suppressed the M current, apparently by acting at a locus downstream from regulation of cAMP levels by adenylate cyclase and phosphodiesterase. -Adrenergic agents may act to increase the number of M channels available to be opened and also modify their kinetics.     

Mutations affecting cyclic phosphodiesterases and adenylate cyclase in Neurospora

Summary Two regulatory mutants in orthophosphateregulated cyclic phosphodiesterase (CPDase), cpd-3 and cpd-4, were isolated and mapped proximal to arg-1 on L.G. IC and distal to urg-12 on L.G. IIR, respectively. cpd-3 showed short aerial hyphae with dense formation of conidia. The morphology was very similar to that of cr-1, cpd-3 and cr-1 had reduced levels of cyclic 3,5-AMP, adenylate cyclase and cPDases (CPDase I, II and III in low phosphate condition) but had elevated levels of cyclic 3,5-GMP. Although cr-1 showed an enhanced level and enhanced activation of heat activated cyclic phosphodiesterase (ha-PDE), this enzyme was not activated in cpd-3. cpd-4, nuc-1 and nuc-2 produced neither CPDase I, II, III, alkaline phosphatase nor ribonuclease N1 in low phosphate media. These mutants did not produce aerial hypha or conidium when grown in low phosphate liquid medium. Activation of ha-PDE occurred in cpd-4, but not in nuc-1 and nuc-2.     

Nitric oxide – cyclic GMP pathway regulates vascular smooth muscle cell phenotypic modulation: implications in vascular diseases

The role of cGMP-dependent protein kinase (PKG) in the regulation of rat aortic vascular smooth muscle cells (VSMC) phenotype was examined using a transfected cell culture system. Repetitively passaged VSMC do not express PKG and exist in the synthetic phenotype. Transfection of PKG-lα cDNA, or the active catalytic domain of PKG-lα, resulted in the appearance of VSMC having a morphology consistent with the contractile phenotype. PKG-expressing cells also contained markers for the contractile phenotype (for example, smooth muscle specific myosin heavy chain, calponin, α-actin) and reduced levels of synthetic phenotype markers (osteopontin, thrombospondin). PKG-transfected VSMC have also reduced the levels of fibroblast growth factor receptors 1 and 2, consistent with the establishment of a more contractile phenotype. The regulation of PKG expression in VSMC is largely undefined; however, continuous exposure of cultured bovine aortic smooth muscle cells with nitric oxide (NO)-donor drugs or cyclic nucleotide analogues reduced the expression of PKG. These results suggest that PKG occupies a critical role in VSMC phenotype and that suppression of PKG expression during inflammation or injury promotes a more synthetic state of the VSMC.     

The Role of Intracellular cAMP in Mediating the Synchronizing Action of Noradrenaline on the Evoked Release of Quanta of Mediator in the Frog Synapse

Experiments on frog neuromuscular junction preparations with extracellular recording of action currents in nerve endings and single-quantum currents from endplates were used to assess the time course of evoked quantum mediator secretion by analyzing histograms showing the distribution of true synaptic delays. Studies using the cyclic AMP analog dibutyryl-cAMP (db-cAMP), the adenylate cyclase activator forskolin, and the nucleotide-dependent phosphodiesterase inhibitor isobutylmethylxanthine, showed that these agents, like noradrenaline, altered the kinetics of secretion of quanta, leading to synchronization of the release of mediator. After preliminary treatment of the neuromuscular preparation with db-cAMP, forskolin, or isobutylmethylxanthine, noradrenaline did not induce the synchronization of mediator release in quanta. It was concluded that the action of noradrenaline on the time course of secretion is mediated by activation of presynaptic receptors, increased adenylate cyclase activity, and increases in intracellular cAMP levels.     

Adenosine as inhibitor of myocardial effects of catecholamines

Summary Infusion of adenosine into the coronary arteries of isolated guinea pig hearts produced a dosedependent inhibition of dP/dt_max caused by bolus injections of isoproterenol (4×10^–11 moles). Threshold concentration of adenosine was 10^–7 M and maximal inhibition (90%) occurred at 10^–5 M. Coronary dilation induced by, papaverine did not influence the contractile response to catecholamines. In addition to its influence on cardiac performance, adenosine (10^–5 M) effectively inhibited the isoproterenol (10^–7 M) induced initial rise in myocardial levels of cyclic 35-AMP, glucose-1-phosphate and glucose-6-phosphate. Adenosine also antagonized the effect of isoproterenol on adenylate cyclase activity in a crude membrane preparation from guinea pig ventricles; it was without effect on the activity of the membrane phosphodiesterase. Theophylline inhibited the actions of adenosine both on adenylate cyclase activity and on contractile force development.-Upon infusion of isoproterenol (3×10^–7 M) into the coronary arteries of the isolated heart (perfusion at constant pressure), the adenosine concentration in the effluent perfusate increased within 45 s from 10^–8 M to about 10^–6 M. It thus appears conceivable that in ventricular myocardium endogenously formed adenosine may serve 2 functions: dilation of the coronary arteries and limitation of the inotropic and metabolic effects of catecholamines.A preliminary report of these studies was presented at the 47th Meeting of the German Physiological Society in Regensburg, Germany [Pflügers Arch.365, R4 (1976)]     

Effect of inhibitors of cyclic nucleotide phosphodiesterases on electrical and contractile activity of smooth muscle cells

Double sucrose gap experiments were carried out to study the effect of phosphodiesterase inhibitors and penetrating analogs of cyclic nucleotides on action potential and contraction of guinea pig ureteral smooth muscle cells. 3-Isobutyl-1-methylxanthine (10 M) and dibutyryl-cAMP (20 M) shortened the plateau of action potential and inhibited contraction of smooth muscle cells by increasing potassium permeability of their membrane. Vinpocetine (1 M) and dibutyryl-cAMP (100 M) strengthened contraction of smooth muscle cells and shortened action potentials by decreasing sodium permeability of their membrane.     

Guanylate-cyclase-mediated inhibition of cardiac I Ca by carbachol and sodium nitroprusside

We studied the role of cyclic guanosine monophosphate (cGMP) as a mediator of the reduction of L-type calcium current (I _Ca) induced by muscarinic receptor stimulation and by nitric oxide in isolated guinea-pig ventricular cells using the whole-cell patchclamp technique. Our results show that when the level of cyclic adenosine monophosphate was increased by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), stimulation of a pertussis-toxin (PTX)-sensitive muscarinic receptor by carbachol (1 M) reduced the calcium current increase from 80.6±23.5% to 19.8±9.6% over the control and this effect was prevented by methylene blue (10 M), an inhibitor of the soluble guanylate cyclase. Pipette solution containing 10 M cGMP reduced the enhancement of I _Ca by IBMX from 121.9±11.6% to 14.2±5.4% above the control. Sodium nitroprusside (10 M), a spontaneous donor of nitric oxide, and consequently a stimulator of soluble guanylate cyclase, also reduced IBMX-stimulated I _Ca from 115.2±13.2% to 32.2±6.9% above control and the sodium nitroprusside effect was also suppressed by methylene blue. The latter two reagents were ineffective on basal I _Ca.     

The response of lymphatic smooth muscles to vasoactive substances

A study was made of the isotonic response of bovine mesenteric lymphatics to several physiological vasoactive substances. Contractions of lymphatic smooth muscles were induced by serotonin (5-HT), prostaglandin F₂ (PGF₂), noradrenaline (NA), histamine, dopamine and acetylcholine (ACh). The smooth muscles were particularly sensitive to 5-HT. Excepting PGF₂ no other substances could equal 5-HT in the magnitude of the maximum response. The majority of 5-HT receptors seemed to be the D receptors. The decreasing order of the contractile responses was as follows: 5-HT>PGF₂>NA>histamine>dopamine>ACh. The contractile response to ACh was observed only in specimens involving valvular region. It was very likely that, in the lymphatics, there were 2 kinds of receptors for catecholamines, i.e. and receptors, and the stimulation of the former induced smooth muscle contraction and that of the latter relaxation. A difference was noticed between the responses of valvular and intervalvular segments to NA. Relaxations of lymphatic smooth muscles were induced not only by isoproterenol but also by adenosine and adenine nucleotides. The decreasing order of the relaxant responses was as follows: ISP>adenosine >ATP>ADP>cyclic AMPAMP. The relaxant responses to adenine nucleotides tended to reduce with decrease in the number of high energy phosphates.     

TVX 2706 — a new phosphodiesterase inhibitor with antiinflammatory action Biochemical characterization

The effects of the anti-inflammatory and analgesic drug 3-ethyl-1-(3-nitrophenyl)-2,4[1H, 3H]-quinazolindione (TVX 2706) on neuronal and glial cell culture systems including neuroblastoma × glioma hybrid cells have been studied. This compound strongly enhances the increase in intracellular levels of cyclic AMP caused by appropriate effectors in all systems tested so far. EC₅₀ values are in the submicromolar range. The effect is apparently neither due to an increased responsiveness of the hybrid cells for an effector like prostaglandin E₁ nor to an increased activity of adenylate cyclase, but to an inhibition of both low and high affinity cyclic AMP phosphodiesterases. Half-maximal inhibition of enzyme activity is obtained at 10 M TVX 2706. The drug is at least equipotent to or more potent than some other common phosphodiesterase inhibitors. Inhibition of phosphodiesterase activity is also observed in homogenates from rat polymorphonuclear leucocytes, where the lowK _m -enzyme is preferentially inhibited. TVX 2706 does not interfere with the calmodulin activation of phosphodiesterase. The role of phosphodiesterase inhibition as a possible mechanism of the anti-inflammatory action of TVX 2706 is discussed.     

Radiation modulation of activity of some enzyme systems of isolated plasma membranes during early ontogeny

Activity of adenylate cyclase (ADCase), cyclic AMP phosphodiesterase (PDE), and 5-nucleotidase was investigated in plasma membranes from the liver of 20-day rat embryos, either normal or exposed to ionizing radiation. Irradiation of the plasma membranes with gamma-rays in doses of between 0.1 and 100 kR was shown to reduce ADCase activity, and to have a more marked effect, in the case of larger doses, when accompanied by stimulation by isoproterenol. The 5-nucleotidase and PDE activity was unchanged under the influence of doses up to 100 kR.Laboratory of Molecular Radiobiology, Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino. (Presented by Academician of the Academy of Medical Sciences of the USSR. P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii Meditsiny, Vol. 89, No. 2, pp. 170–172. February, 1980.     

Inhibition of cyclic AMP phosphodiesterase of the rat heart and brain by neurohormone C in vitro

The effect of neurohormone C, a new coronary dilator isolated from bovine hypothalamus, on activity of the phosphodiesterase (PDE) of cyclic adenosine-3,5-monophosphate (AMP) was investigated. Considerable inhibition of PDE was shown in the supernatant of rat brain and heart homogenates (2000g) under the influence of neurohormone C. The existence of correlation between the coronary dilator effect of the hormone and regulation of the cyclic AMP level under the influence of a change in PDE activity is postulated.Laboratory of Hormones, Institute of Biochemistry, Academy of Sciences of the Armenian SSR, Erevan. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 6, pp. 691–693, June, 1977.     

Defective leukocyte adenylate cyclase function in hypokalemia

Summary Patients with hypokalemia due to Bartter's syndrome show an increase in adrenergic nervous system function with significantly elevated plasma norepinephrine excretion. Prolonged exposure to neurotransmitters or hormones is known to lead to a down-regulation of target-cell responsiveness. We measured cyclic AMP generation by leukocytes in response to the -adrenergic agonist isoproterenol and to prostaglandin E₁ (PGE₁) in six patients with Bartter's syndrome. As compared to normal controls, the response of cyclic AMP production by leukocytes to stimulation by 1-isoproterenol or PGE₁ was significantly decreased. These results indicate that in Bartter's syndrome and probably in other diseases involving hypokalemia isoproterenol- and PGE₁-sensitive adenylate cyclase activities of leukocytes are reduced. Because the effect of PGE₁ on adenylate cyclase is not mediated through the specific -adrenoceptor, it is possible that a defect in receptor-adenylate cyclase coupling or a more distal post-receptor defect is responsible for the reduction in cyclic AMP production.Abbreviations cyclic AMP adenosine 3:5 - cyclic monophosphate Portions of this work have been presented at the Annual Meeting of the American Society for Clinical Investigation, April 29 to May 2, 1983, Washington, DC (Clin Res 1983; 31:471A)