Multiple classes of dopamine receptors in mammalian central nervous system: the involvement of dopamine-sensitive adenylyl cyclase.

Two classes of dopamine receptor mechanism are defined according to their association with, or independence from, a dopamine-sensitive adenylyl cyclase. Dopamine receptors unrelated to adenylyl cyclase are designated type alpha. Dopamine receptors linked to adenylyl cyclase are designated type beta. Drugs discriminate between the two receptor mechanisms. The dopaminergic ergots (lisuride, lergotrile and CB-154) and their antagonists (such as metoclopramide) are relatively specific for the alpha-dopaminergic receptor in the anterior pituitary. Other agonists (e.g. apomorphine and dopamine) and antagonists (e.g. antipsychotic phenothiazines and butyrophenones) affect both classes of receptor.     

Regulation of adenylyl cyclase isoforms by N-alkanols

We examined the effect of n-alkanols on adenylyl cyclase isoforms (types II and V) overexpressed in insect cells. Ethanol stimulated the type II isoform but not the type V isoform. Ethanol stimulated type II adenylyl cyclase greater than GTPγS, and the treatment of the membrane with GDPβS or cholera toxin did not affect this stimulation. Other n-alkanols inhibited type V adenylyl cyclase activity in proportion to their lipophilic potency. In contrast, type II adenylyl cyclase was stimulated by weakly lipophilic n-alkanols and inhibited by strongly lipophilic n-alkanols. When solubilized membranes and purified preparations were used, all the n-alkanols inhibited type II adenylyl cyclase. Our data suggest that n-alkanols regulated adenylyl cyclase isoform-dependently. Stimulation of the type II isoform was independent from the interaction with Gsα but required the presence of an intact membrane structure. Our study may provide another step to understanding how membrane protein subtypes are differentially regulated by n-alkanols. J. Cell. Biochem. 66:450–456, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells

We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the β-adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G-protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G-proteins, adenylyl cyclase, and phosphodiesterase.     

An octopamine-sensitive adenylate cyclase in the central nervous system of Limulus polyphemus

• 1.1. Adenylate cyclase (E.C. 4.6.1.1) was assayed and shown to be present in the protocerebrum and circumesophageal ring of Limulus polyphemus.
• 2.2. The enzyme activity from both tissues is stimulated by fluoride and guanylnucleotides.
• 3.3. The circumesophageal ring, but not the protocerebrum, is responsive to octopamine.
• 4.4. Octopamine stimulation of the adenylate cyclase is reversed by phentolamine and dopamine.

     

Regulation of proliferation during central nervous system development

The retina is one of the best-characterized regions of the central nervous system (CNS) and has served as a model for many of the principles that now form the foundation for CNS development. In the past several years, a number of advances have been made in our understanding of the coordination of proliferation and cell fate specification during retinal development. In this review, we will draw on findings from studies of the retina and highlight similarities and differences in other regions in the CNS, namely the cerebellum and cortex. We will present a framework in which to pose challenges and outstanding questions for future studies on the coordination of proliferation and cell fate specification in the developing CNS.     

Regulation of cytochrome P450 in the central nervous system

The role of brain P450 in the physiology, pharmacology and toxicology of the brain is the subject of this study. Cytochrome P450 was isolated from the brains of rats and quantitated spectrally. The contribution of the known hepatic forms of the enzyme to the forms constitutive in the brain as well as those which are induced by hormones are xenobiotics were characterized on Western blots. We have found that the level of P450 in the brain is increased during pregnancy and lactation, by partial hepatectomy and by ethanol. In each case the profile of P450s induced is different. In pregnancy and lactation the P450 content of the hypothalamic preoptic area and olfactory lobes were increased up to 10-fold and the only subfamily identified on Western blots was 4A. There was no detectable 1A, 2A, 2B, 2C, or 2E1. Ethanol increases the level of brain P450 3- to 5-fold and P450 2C, 2E1 and 4A are induced. Upon partial hepatectomy P450 1A, 2C and 4A were detected on Western blots but there was no 2E1. The inducibility of these forms of P450 in the brain suggests that there is in situ metabolism of steroids, fatty acids, prostaglandins, ethanol and other xenobiotics in the brain and raises questions about the role of brain P450 in the development of tolerance to drugs and the neurotoxicity of xenobiotics. More importantly, the action of neurotransmitters such as dopamine which utilize fatty acid metabolites as intracellular mediators, could be influenced by the levels of 2C and 4A P450s.     

Regulation of bone remodeling by the central and peripheral nervous system

The homeostatic nature of bone remodeling has become a notion further supported lately by the demonstration that neuropeptides and their receptors regulate osteoblast and osteoclast function in vivo. Following initial studies reporting the presence of nerves and nerve-derived products within the bone microenvironment and the expression of receptors for these neuropeptides in bone cells, new experimental and mechanistic evidence based on in vivo murine genetic and pharmacologic models recently demonstrated that inputs from the central and peripheral nervous system feed into the already complex regulatory machinery controlling bone remodeling. The function of a number of “osteo-neuromediators” has been characterized, including norepinephrine and the beta2-adrenergic receptor, Neuropeptide Y and the Y1 and Y2 receptors, endocannabinoids and the CB1 and CB2 receptors, as well as dopamine, serotonin and their receptors and transporters, Calcitonin gene-related peptide, and neuronal NOS. This new body of evidence suggests that neurons in the central nervous system integrate clues from the internal and external milieux, such as energy homeostasis, glycemia or reproductive signals, with the regulation of bone remodeling. The next major tasks in this new area of bone biology will be to understand, at the molecular level, the mechanisms by which common central neural systems regulate and integrate these major physiological functions, the relative importance of the central and peripheral actions of neuropeptides present in both compartments and their relationship, and how bone cells signal back to central centers, because the definition of a homeostatic function implies the existence of feedback signals. Together, these findings shed a new light on the complexity of the mechanisms regulating bone remodeling and uncovered new potential therapeutic strategies for the design of bone anabolic treatments. This review summarizes the latest advances in this area, focusing on investigations based on in vivo animal studies.     

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.     

The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.     

Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body

Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.     

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca²⁺/phospholipid-dependent protein kinase, and Ca²⁺/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.     

Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system

The nuclear oxysterol receptors liver X receptor-alpha [LXRalpha (NR1H3)] and LXRbeta (NR1H2) coordinately regulate genes involved in cholesterol homeostasis. Although both LXR subtypes are expressed in the brain, their roles in this tissue remain largely unexplored. In this report, we show that LXR agonists have marked effects on gene expression in murine brain tissue both in vitro and in vivo. In primary astrocyte cultures, LXR agonists regulated several established LXR target genes, including ATP binding cassette transporter A1, and enhanced cholesterol efflux. In contrast, little or no effect on gene expression or cholesterol efflux was detected in primary neuronal cultures. Treatment of mice with a selective LXR agonist resulted in the induction of several LXR target genes related to cholesterol homeostasis in the cerebellum and hippocampus. These data provide the first evidence that the LXRs regulate cholesterol homeostasis in the central nervous system. Because dysregulation of cholesterol balance is implicated in central nervous system diseases such as Alzheimer's and Niemann-Pick disease, pharmacological manipulation of the LXRs may prove beneficial in the treatment of these disorders.     

Regulation of type I adenylyl cyclase by calmodulin kinase IV in vivo.

Type I adenylyl cyclase is a neurospecific enzyme that is stimulated by Ca2+ and calmodulin (CaM). This enzyme couples the Ca2+ and cyclic AMP (cAMP) regulatory systems in neurons, and it may play an important role for some forms of synaptic plasticity. Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (Z. Wu, S. A. Thomas, Z. Xia, E. C. Villacres, R. D. Palmiter, and D. R. Storm, Proc. Natl. Acad. Sci. USA 92:220-224, 1995). Although type I adenylyl cyclase is synergistically stimulated by Ca2+ and G-protein-coupled receptors in vivo, very little is known about mechanisms for inhibition of the enzyme. Here, we report that type I adenylyl cyclase is inhibited by CaM kinase IV in vivo. Expression of constitutively active or wild-type CaM kinase IV inhibited Ca2+ stimulation of adenylyl cyclase activity without affecting basal or forskolin-stimulated activity. Type I adenylyl cyclase has two CaM kinase IV consensus phosphorylation sequences near its CaM binding domain at Ser-545 and Ser-552. Conversion of either serine to alanine by mutagenesis abolished CaM kinase IV inhibition of adenylyl cyclase. This suggests that the activity of this enzyme may be directly inhibited by CaM kinase IV phosphorylation. Type VIII adenylyl cyclase, another enzyme stimulated by CaM, was not inhibited by CaM kinase II or IV. We propose that CaM kinase IV may function as a negative feedback regulator of type I adenylyl cyclase and that CaM kinases may regulate cAMP levels in some cells.     

Identity of adenylyl cyclase isoform determines the G protein mediating chronic opioid-induced adenylyl cyclase supersensitivity

To determine the intracellular signal transduction pathway responsible for the development of tolerance/dependence, the ability of Gzalpha to substitute for pertussis toxin (PTX)-sensitive G proteins in mediating adenylyl cyclase (AC) supersensitivity was examined in the presence of defined AC isoforms. In transiently micro-opioid receptor (OR) transfected COS-7 cells (endogenous inhibitory G proteins: Gialpha2, Gialpha3 and Gzalpha), neither acute (1 micro mol/L) nor chronic morphine treatment (1 micromol/L; 18 h) influenced intracellular cAMP production. Coexpression of the micro -OR together with AC type V and VI fully restored the ability of morphine to acutely inhibit cAMP generation. Chronic morphine treatment further resulted in the development of tolerance/dependence, as assessed by desensitization of the acute inhibitory opioid effect (tolerance) as well as the induction of AC supersensitivity after drug withdrawal (dependence). Specific direction of micro -OR signalling via Gzalpha by both PTX treatment and Gzalpha over-expression had no effect on chronic morphine regulation of AC type V, but completely abolished the development of tolerance/dependence with AC type VI. Similar results were obtained in stably micro -OR-expressing HEK293 cells transiently cotransfected with Gzalpha and either AC type V or VI. Coprecipitation studies further verified that Gzalpha specifically binds to AC type V but not type VI. Taken together, these results demonstrate that in principle each of the OR-activated G proteins per se is able to mediate AC supersensitivity. However, they also indicate that it is the molecular nature of AC isoform that selects and determines the OR-activated G protein mediating tolerance/dependence.     

The adenylyl cyclase family

Hormone-sensitive adenylyl cyclase is a model system for the study of receptor-mediated signal transduction. It is comprised of three types of components: 1) receptors for hormones that regulate cyclic AMP (cAMP) synthesis, 2) regulatory GTP binding proteins (G proteins), and 3) the family of enzymes, the adenylyl cyclases. Concentrations of cAMP are altered by at least 35 different stimulatory or inhibitory hormones and neurotransmitters. Other signalling pathways may also influence cAMP production through regulation of particular adenylyl cyclase subtypes. The second messenger, cAMP propagates the hormone signal through the effects of cAMP-dependent protein kinase.While structural information on the adenylyl cyclases is limited, a cDNA clone for a calmodulin-sensitive form of bovine brain adenylyl cyclase has been isolated. The amino acid sequence encoded by the Type I cDNA is approximately 40% identical to those specified by three other adenylyl cyclase cDNAs that have been cloned subsequently. This degree of structural variation implies that there must be functional differences between the adenylyl cyclases.     

Endocannabinoids in the central nervous system

The major psychoactive component of cannabis derivatives, delta9-THC, activates two G-protein coupled receptors: CB1 and CB2. Soon after the discovery of these receptors, their endogenous ligands were identified: lipid metabolites of arachidonic acid, named endocannabinoids. The two major main and most studied endocannabinoids are anandamide and 2-arachidonyl-glycerol. The CB1 receptor is massively expressed through-out the central nervous system whereas CB2 expression seems restricted to immune cells. Following endocannabinoid binding, CB1 receptors modulate second messenger cascades (inhibition of adenylate cyclase, activation of mitogen-activated protein kinases and of focal-adhesion kinases) as well as ionic conductances (inhibition of voltage-dependent calcium channels, activation of several potassium channels). Endocannabinoids transiently silence synapses by decreasing neurotransmitter release, play major parts in various forms of synaptic plasticity because of their ability to behave as retrograde messengers and activate non-cannabinoid receptors (such as vanilloid receptor type-1), illustrating the complexity of the endocannabinoid system. The diverse cellular targets of endocannabinoids are at the origin of the promising therapeutic potentials of the endocannabinoid system.