Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands

Levels of serotonin N-acetyltransferase (NAT: acetul CoA:arylamine N-acetyltransferase; EC 2.1.1.5.) activity in the chick pineal gland exhibit a marked diurnal variation in birds kept under a diurnal cycle of ilumination. Activity begins to rise rapidly at the start of the dark phase of the cycle and reaches maximum levels at mid-dark phase about 25-fold greater than the minimum basal level at mid-light phase. Thereafter, the level of activity declines to the basal level about the start of the light phase. This diurnal cycle in chick pineal NAT activity found in vivo has recently been reproduced in vitro with intact glands incubated in organ culture. The mechanism of the 'biological clock' which regulates these variations in level of chick pineal NAT activity is unknown. However, I now report that chick pineal glands cultured under a diurnal cycle of illumination exhibit a diurnal cycle in content of cyclic GMP which roughly parallels the cycles in NAT activity. In contrast, there was no correlation between variations in pineal content of cyclic AMP and in level of NAT activity.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

A cyclic GMP-activated channel in dissociated cells of the chick pineal gland.

Phototransduction in the vertebrate retina is dependent in part on a cyclic GMP-activated ionic channel in the plasma membrane of rods and cones. But other vertebrate cells are also photosensitive. Cells of the chick pineal gland have a photosensitive circadian rhythm in melatonin secretion that persists in dissociated cell culture. Exposure to light causes inhibition of melatonin secretion, and entrainment of the intrinsic circadian oscillator. Chick pinealocytes express several 'retinal' proteins, including arrestin, transducin and a protein similar to the visual pigment rhodopsin. Pinealocytes of lower vertebrates display hyperpolarizing responses to brief pulses of light. Thus it is possible that some of the mechanisms of phototransduction are similar in retinal and pineal photoreceptors. We report here the first recordings of cyclic GMP-activated channels in an extraretinal photoreceptor. Application of GMP, but not cyclic AMP, to excised inside-out patches caused activation of a 15-25 pS cationic channel. These channels may be essential for phototransduction in the chick pineal gland.     

Phosducin is a protein kinase A-regulated G-protein regulator.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.     

Beta-adrenergic modulation of calcium channels in frog ventricular heart cells

Adrenergic modulation of calcium channels profoundly influences cardiac function, and has served as a prime example of neurohormonal regulation of voltage-gated ion channels. Channel modulation and increased Ca influx are mediated by elevation of intracellular cyclic AMP and protein phosphorylation. The molecular mechanism of the augmented membrane Ca conductance has attracted considerable interest. An increase in the density of functional channels has often been proposed, but there has previously been no direct evidence. Single-channel recordings show that isoprenaline or 8-bromocyclic AMP increase the proportion of time individual channels spend open by prolonging openings and shortening the closed periods between openings. To look for an additional contribution of changes in the number of functional channels, we applied ensemble fluctuation analysis to whole-cell recordings of cardiac Ca channel activity. Here we present evidence that in frog ventricular heart cells beta-adrenergic stimulation increases NF, the average number of functional Ca channels per cell. We also find that isoprenaline slows the time course of both activation and inactivation, and that the enhancement of peak current decreases gradually with greater membrane depolarization.     

Activation of facilitation calcium channels in chromaffin cells by D1 dopamine receptors through a cAMP/protein kinase A-dependent mechanism

Facilitation calcium channels in unstimulated bovine chromaffin cells are normally quiescent but are activated by large pre-depolarizations or by repetitive depolarization in the physiological range. The activation of these 27-pS dihydropyridine-sensitive channels by repetitive stimulation, such as by increased splanchnic nerve activity, can lead to an almost twofold increase in Ca2+ current in these cells. This increase in Ca2+ current is of probable physiological importance in stimulating rapid catecholamine secretion in response to danger or stress. We have identified D1 dopaminergic receptors on bovine chromaffin cells by fluorescence microscopy. Here we show that stimulation of the D1 receptors activates the facilitation Ca2+ currents in the absence of pre-depolarizations or repetitive activity, and that activation by D1 agonists is mediated by cyclic AMP and protein kinase A. The recruitment of facilitation Ca2+ channels by dopamine may form the basis of a positive feedback loop mechanism for catecholamine secretion.     

Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes

Hormones mobilize intracellular second messengers and initiate signalling cascades involving protein kinases and phosphatases, which are often spatially compartmentalized by anchoring proteins to increase signalling specificity. These scaffold proteins may themselves be modulated by hormones. In adipocytes, stimulation of beta-adrenergic receptors increases cyclic AMP levels and activates protein kinase A (PKA), which stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. Acute insulin treatment activates phosphodiesterase 3B, reduces cAMP levels and quenches beta-adrenergic receptor signalling. In contrast, chronic hyperinsulinaemic conditions (typical of type 2 diabetes) enhance beta-adrenergic receptor-mediated cAMP production. This amplification of cAMP signalling is paradoxical because it should enhance lipolysis, the opposite of the known short-term effect of hyperinsulinaemia. Here we show that in adipocytes, chronically high insulin levels inhibit beta-adrenergic receptors (but not other cAMP-elevating stimuli) from activating PKA. We measured this using an improved fluorescent reporter and by phosphorylation of endogenous cAMP-response-element binding protein (CREB). Disruption of PKA scaffolding mimics the interference of insulin with beta-adrenergic receptor signalling. Chronically high insulin levels may disrupt the close apposition of beta-adrenergic receptors and PKA, identifying a new mechanism for crosstalk between heterologous signal transduction pathways.     

Noradrenaline and cyclic AMP--independent growth stimulation in newt limb blastemata

Adult newts regenerate functional limbs after amputation. This process normally depends on the trophic influence of nerves on the regenerating limbs, particularly in the early stages before differentiation of the regeneration blastema, when it stimulates growth by maintaining high rates of macromolecular synthesis. The sequence of biochemical events involved is unknown, but it has been suggested that intracellular cyclic AMP may be a second messenger within the blastema. Many studies have indicated that the neural agent(s) involved might be protein. The recent finding that blastemata contain high levels of catecholamines, however, has implicated noradrenaline (NA) as the neurotrophic agent, and suggested that it works via stimulation of beta-adrenergic receptors on the blastemal cells, thereby raising the intracellular concentrations of cyclic AMP. To test this hypothesis we studied the ability of NA alone and in combination with alpha-and beta-adrenergic antagonists to increase cyclic AMP levels and to mimic the effects of nerves by maintaining high rates of protein synthesis and high mitotic indices (MI) in denervated blastemata in organ culture. We find that although NA raises cyclic AMP levels through a beta-adrenergic effect, it does not maintain high rates of protein synthesis or high MI in cultured blastemata. It is unlikely therefore, that this hypothesis applies.     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.     

Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins.

Many hormones, neurotransmitters and growth factors, on binding to G protein-coupled receptors or receptors possessing tyrosine kinase activity, increase intracellular levels of the second messengers inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This is due to activation of phosphoinositide-specific phospholipase(s) C (PLC), the isozymes of which are classified into groups, alpha, beta, gamma and delta. The beta, gamma and delta groups themselves contain PLC isozymes which have both common and unique structural domains. Only the gamma 1 isozyme has been implicated in a signal transduction mechanism. This involves association with, and tyrosine phosphorylation by, the ligand-bound epidermal growth factor and platelet-derived growth factor receptors, probably by means of the PLC-gamma 1-specific src homology (SH2) domain. Because EGF receptor-mediated tyrosine phosphorylation of PLC-gamma 1 stimulates catalytic activity in vitro and G proteins have been implicated in the activation of PLC, we investigated which PLC isozymes are subject to G protein regulation. We have purified an activated G protein alpha subunit that stimulates partially purified phospholipase C and now report that this G protein specifically activates the beta 1 isozyme, but not the gamma 1 and delta 1 isozymes of phospholipase C. We also show that this protein is related to the Gq class of G protein alpha subunits.     

Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.     

Cross-talk between cellular signalling pathways suggested by phorbol-ester-induced adenylate cyclase phosphorylation

Receptor-mediated activation of both adenylate cyclase and phosphatidylinositide hydrolysis systems occurs through guanine nucleotide regulatory proteins and ultimately leads to specific activation of either cyclic AMP-dependent protein kinase A or Ca2+/phospholipid-dependent protein kinase C. Given the remarkable diversity of agents that influence cellular metabolism through these pathways and the similarities of their components, interactions between the two signalling systems could occur. In fact, stimulation of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a phorbol ester that activates protein kinase C, influences hormone-sensitive adenylate cyclase. In some cells TPA induces desensitization of receptor-mediated stimulation of adenylate cyclase, whereas in others, such as frog erythrocytes, phorbol ester treatment results in increased agonist-stimulated as well as basal, guanine nucleotide- and fluoride ion-stimulated adenylate cyclase activities. We show here that TPA produces phosphorylation of the catalytic unit of adenylate cyclase in frog erythrocytes. Moreover, purified protein kinase C can directly phosphorylate in vitro the catalytic unit of adenylate cyclase purified from bovine brain. These results suggest that phosphorylation of the catalytic unit of adenylate cyclase by protein kinase C may be involved in the phorbol ester-induced enhancement of adenylate cyclase activity. In addition to providing the first direct demonstration of a covalent modification of the catalytic unit of adenylate cyclase, these results provide a potential biochemical mechanism for a regulatory link between the two major transmembrane signalling systems.     

Stimulation of p21ras upon T-cell activation

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.     

Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2

Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.     

An isoprenaline activated sodium-dependent inward current in ventricular myocytes

In the heart, catecholamines affect pacemaker activity by shifting the activation curve for the nonspecific inward current and increasing both the calcium current, and the delayed potassium current. We report here that in mammalian ventricle there is another mechanism that seems to involve a sodium-dependent inward current. This is elicited by agents that increase intracellular cyclic AMP concentration, such as the beta-adrenergic agonist isoprenaline, and is unaffected by agents which block the three currents listed above, but is absent when external sodium is replaced with tetramethylammonium. Most interestingly, the intracellular pathway(s) linking the beta-receptor(s) to activation of the Ca current and the Na-dependent current, which in both cases presumably involves the intracellular concentration of cAMP, differ, as isoprenaline causes a persistent augmentation of the calcium current whereas the Na-dependent current often fades. These effects of isoprenaline are antagonized by acetylcholine. In unclamped cells, the Na-dependent current depolarizes the membrane to the potential range at which repetitive firing occurs. It may therefore be involved in the generation of ventricular arrhythmias.     

Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells

The cell-to-cell permeability of the junctions of various cultured mammalian cell types depends on the concentration of intracellular cyclic AMP [( cAMP]i). The permeability rises when the cells are supplied with exogenous cyclic AMP or when their cyclic AMP synthesis is stimulated with choleragen or hormones; it falls when [cAMP]i is lowered by application of serum or due to increase in cell density. The rise and fall in permeability take several hours to develop (the rise is protein synthesis-dependent) and they occur concurrently with the rise and fall in the number of intramembrane particles of the gap junctions, which probably embody the cell-to-cell channels. Is this permeability regulation mediated by phosphorylating protein kinase? In many eukaryotes, the cyclic AMP receptor is a protein kinase consisting of a pair of regulatory subunits and a pair of catalytic subunits. The latter dissociate from the holoenzyme as the cyclic AMP binds to the regulatory subunits and, in this dissociated form, catalyse the phosphorylation of the target. The regulatory subunit occurs in two isoenzyme forms, I and II. The catalytic subunit seems invariant; subunits from different isoenzymes can substitute for each other. We show here that a mutant cell lacking the isoenzyme I is deficient in permeable junctions, and that this junctional defect is corrected when the mutant is supplied with exogenous catalytic subunit.     

Neuropeptides modulate the beta-adrenergic response of purified astrocytes in vitro

Neuropeptides may have functions in the central nervous system (CNS) other than altering neuronal excitability. For example, they may act as regulators of brain metabolism by affecting glycogenolysis. Since it has been suggested that glial cells might provide metabolic support for neuronal activity, they may well be one of the targets for neuropeptide regulation of metabolism. Consistent with this view are reports that peptide-containing nerve terminals have been seen apposed to astrocytes, but it is also quite possible that peptides could act at sites lacking morphological specialization. Primary cultures containing CNS glial cells have been shown to respond to beta-adrenergic agonists with an increase in cyclic AMP and, as a result, with an increase in glycogenolysis and have also been shown to respond to a variety of peptides with changes in cyclic AMP. In the study reported here, we have examined the effects of several peptides on relatively pure cultures of rat astrocytes. We demonstrate that the increase in intracellular cyclic AMP induced by noradrenaline is markedly enhanced by somatostatin and substance P and is inhibited by enkephalin, even though these peptides on their own have little or no effect on the basal levels of cyclic AMP. Vasoactive intestinal peptide (VIP) on the other hand increases cyclic AMP in the absence of noradrenaline. These results suggest that neuropeptides influence glial cells as well as neurones in the CNS and, in the case of somatostatin and substance P, provide further examples of neuropeptides modulating the response to another chemical signal without having a detectable action on their own.     

Tissue-specific expression of three distinct types of rabbit protein kinase C

We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.     

Chronic ethanol causes heterologous desensitization of receptors by reducing alpha s messenger RNA

One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.