Extracellular Messengers Research Articles

Calcium regulation in vertebrates: An overview

All cells maintain the calcium concentration of their cytoplasm below that of the surrounding fluids. In resting eucaryotic cells the cytoplasmic calcium activity is less than 1 /*M and although there have been no direct measurements in procaryotes, indirect evidence suggests that here too it may be less than 1 PM (Ordal, 1977). Why should Ca2+ be maintained at such a low concentration m all cells? The answer may ultimately derive from the very low solubility of calcium phosphates in water. The phosphatedominated energy economy of cells is of very ancient origin and, at neutral pH, is incompatible with high concentrations of calcium (Kretsinger, 1977). The maintenance of a low intracellular calcium concentration must, therefore, have long been of paramount importance. Cells have the capacity to regulate their internal calcium concentration and, in many cases, they can restore it to normal after periods of rapid calcium entry. It, therefore, seems unlikely that the resting cytoplasmic calcium activity would be directly affected by modest changes in the calcium concentration of extracellular fluids. Nevertheless, it is unclear to what extent precise intracellular calcium homeostasis is dependent on a stable extracellular calcium concentration (Bronner, 1982). In mammals, even small changes in plasma calcium concentration can be deleterious. Hypocalcaemia, for example, causes neuromuscular hyperexcitabihty which seems to result from an extracellular effect of calcium on membrane resistance (Frankenhaeuser and Hodgkin, 1957). Changes in extracellular calcium concentration also substantially affect the responses of cells to agonists that gate calcium, for example, glucosestimulated insulin release from pancreatic islets (Curry et al., 1968). Although the resting cell may be able to regulate its cytoplasmic calcium activity despite changes in plasma calcium, the extracellular and second messenger functions of calcium require that the plasma calcium concentration is stable. For some marine invertebrates that stability is provided by a

A cDNA clone encoding neuropeptides isolated from Aplysia neuron L11.

Single nerve cells can use more than one substance as extracellular chemical messengers. Classical transmitters have been shown to coexist in the same neuron and possibly even in the same vesicle as neuroactive peptides. Furthermore, multiple neuroactive peptides, which are thought to be coreleased, are often encoded in the same precursor assuring stoichiometric synthesis. The precise organization of multiple message systems and the physiological significance of the coexistence is poorly understood. The abdominal ganglion of the gastropod mollusc Aplysia contains a number of identified neurons that are cotransmitter candidates. One such cell, L11, is cholinergic and probably also uses biologically active peptides. Differential screening with labeled cDNA was used to isolate cDNA clones expressed specifically in the bag cells and abdominal ganglion neurons L11 or R15. Analysis of an L11-specific clone suggests that it encodes a 14.7-kDa protein that is the precursor for the secreted peptides. The poly(A)+ RNA transcript is approximately equal to 1.2 kilobases and there are 1-3 copies of this gene in the Aplysia haploid genome.

Solubilized active pituitary and ovarian gonadotropin-releasing hormone receptors retain binding properties for adenosine 3′, 5′-cyclic monophosphate derivatives

The non-denaturing zwitterionic detergent, (3 (3-cholamidopropyl)-dimethyl-ammonio)-1-propane sulfonate (CHAPS), has been used to solubilize membrane gonadotropin-releasing hormone (GnRH) receptors from rat ovaries. The solubilized receptors retain a high affinity (K a = 1.85 ± 0.3 nM −1), comparable to the affinity measured in membrane particles (K a = 3.25 ± 0.7 nM −1), and a preserved specificity for several analogs and fragments of GnRH. At millimolar concentrations, cyclic AMP derivatives inhibit [125 I] - GnRH analog binding to both membrane particles and soluble receptors from pituitary and ovary. These results support the hypothesis that cyclic AMP may play the role of an extracellular messenger by interacting with the GnRH receptor itself.

Receptors and recognition: from ligand binding to gene structure

The concept of recognition of extracellular messengers by membrane-bound or internal molecules which translate ligand binding into cell activation has, in the last decade, led to striking new insights into the structure and function of receptors. It is beyond the scope of this article to list all the successes which have been attained in the isolation of receptors and the characterization of their structure and function. Instead I shall summarize the general facts about molecular recognition which are now established and list some of the questions that remain to be answered.

The insulin receptor

Cells are endowed with specific cognitive molecules that function as receptors for hormones, neurotransmitters, and other intercellular messengers. The receptor molecules may be present in the plasma membrane, cytoplasm, or nucleus. When occupied by the messenger, the receptor is coupled to the cellular machinery that responds to the message-bearing molecules. For some hormones the events following attachment of the messenger to the receptor are well known. An example is the generation of cAMP after combination of glucagon with its receptor and the series of steps culminating in activation of phosphorylase. In the case of many other messengers, including insulin, the nature of these coupling steps is not known. Receptors are subject to the regulatory processes of synthesis, degradation, and conformational change; alterations in receptor properties may have significant effects on the qualitative and quantitative responses of the cell to the extracellular messenger. The insulin receptor is located in the plasma membrane, is composed of two pairs of subunits, and has a molecular weight of about 350,000. It is located in cells such as adipocytes, hepatocytes, and skeletal muscle cells as well as in cells not considered to be typical target organ cells. Insulin receptors in nonfetal cells are downregulated by exposure of the cells to high concentrations of insulin. Other factors that regulate insulin binding include muscular exercise, diet, thyroid hormones, glucocorticoids, androgens, estrogens, and cyclic nucleotides. The fetus has high concentrations of insulin receptors in several tissues. These begin to appear early in fetal life and may outnumber those found in adult tissues. Fetal insulin receptors are unusual in that they may not undergo downregulation but may experience the opposite when exposed to insulin in high concentrations. Thus the offspring of a mother with poorly controlled diabetes may be placed in double jeopardy by fetal hyperinsulinemia and augmented insulin binding by the receptors. Many disorders in children and adults are associated with changes in the properties of the insulin receptor. In general, the alterations have been measured in receptor-bearing cells that are readily accessible, such as circulating monocytes and erythrocytes. The receptors on these cells generally reflect the status of receptors on the major target organs of insulin, although exceptions are known, and conclusions drawn from studies of receptors on circulating cells must be made with caution.(ABSTRACT TRUNCATED AT 400 WORDS)

Synergistic functions of protein phosphorylation and calcium mobilization in platelet activation.

When human platelets were stimulated by synthetic diacylglycerol such as 1-oleoyl-2-acetyl-glycerol, which was a potent activator in vitro of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) (Mori, T., Takai, Y., Yu, B., Takahashi, J., Nishizuka, Y., and Fujikura, T. (1982) J. Biochem. (Tokyo) 91, 427-431), a protein having Mr approximately 40,000 (40-kilodalton protein) was rapidly phosphorylated, just as it was by natural extracellular messengers such as thrombin. Fingerprint analysis appeared to indicate that protein kinase C was indeed responsible for this 40-kilodalton protein phosphorylation in intact platelets. Under these conditions, neither inositol phospholipid breakdown nor endogenous diacylglycerol formation was observed, indicating that the synthetic diacylglycerol intercalated into the membrane and directly activated protein kinase C without interaction with cell surface receptors. During this process, the diacylglycerol was converted in situ to the corresponding phosphatidate, 1-oleoyl-2-acetyl-glyceryl-3-phosphoric acid. Experiments with the synthetic diacylglycerol and Ca2+ ionophore A23187 suggested that the protein phosphorylation catalyzed by protein kinase C was a prerequisite requirement for the release of serotonin, and that the receptor-linked protein phosphorylation and Ca2+ mobilization acted synergistically to elicit the full physiological cellular response.

26] Ca2+-dependent neutral protease and proteolytic activation of Ca2+-activated, phospholipid-dependent protein kinase

Publisher Summary This chapter focuses on the purification and properties of the rat brain Ca2+-protease I and the nature of the proteolytic activation of protein kinase C. It also presents some of the properties of Ca2+-protease II comparison. This protease-protein kinase interaction may provide not only a specific and sensitive assay for this group of proteases, but also a clue for exploring biological roles of the proteolytic activation of this potentially multifunctional protein kinase. The protein kinase is apparently capable of mediating the information of a variety of extracellular messengers across cell membranes. Protein kinase C is normally inactive, but is activated by association with membrane phospholipid in the presence of the micromolar range of Ca2+. This activation process is reversible and is initiated by diacylglycerol, which is derived from the receptor-linked breakdown of phosphatidylinositol in such a way that diacylglycerol greatly increases the affinity of enzyme for Ca2+, as well as for phospholipid. Ca2+protease I reacts preferentially with the phospholipid-associated form of protein kinase C and produces an enzyme fragment. This enzyme fragment is enzymically fully active without Ca2+, phospholipid, and diacylgycerol. In the absence of phospholipid, protein kinase C is far less susceptible to Ca2+-protease I.

Chemotaxis in Dictyostelium

Dictyostelium discoideum exists as single amoeboid cells during the first phase of its developmental cycle. These cells phagocytose bacteria. Chemo­ taxis to compounds released from the bacteria (e.g. folic acid) is probably involved in food seeking (95). After the growth phase the amoebae differen­ tiate into cells capable of aggregating into a multicellular organism. A breakthrough in the investigation of Dictyostelium chemotaxis was the discovery in 1967 that aggregating cells of D. discoideum are attracted by cyclic AMP (cAMP) (64). Normally in the laboratory D. discoideum devel­ ops asexually, but by combining cells of different mating types sexual devel­ opment can be induced. Chemotaxis is also involved in sexual development as the cells are attracted by immature macrocysts (93). These are cell groups in which zygotes are formed. In the multicellular stage the cells differentiate into spores and cells forming a stalk and basal disk. These two types of cells constitute the fruiting body formed at the end of development. Evidence has been presented for an action of cAMP in the sorting out of pre-spore and pre-stalk cells, suggesting that the spatial pattern of these cells is determined by chemotaxis (88). Chemotaxis to oxygen seems also to be involved in aligning the pre-spore/pre-stalk pattern in the stage preceding fruiting body formation (114). The discovery that cAMP is a chemoattractant established cAMP as a primary, extracellular messenger. Cyclic AMP exerts its function as a che­ moattractant by binding to cell-surface receptors (47, 49, 70, 78). In addi­ tion, cAMP affects cell differentiation (16, 18, 35) and stimulates its own

Phosphatidylinositol turnover in platelet activation; Calcium mobilization and protein phosphorylation

Ca2+-activated, phospholipid-dependent protein kinase (C-kinase) in platelets is normally activated by diacylglycerol, which is derived from phosphatidylinositol through its receptor-linked breakdown. Under appropriate conditions this enzyme can also be activated by synthetic diacylglycerol which is directly added to intact platelets. C-Kinase thus activated preferentially phosphorylates an endogenous platelet protein having a molecular weight of approximately 40,000. This protein phosphorylation is merely a prerequisite but not a sufficient requirement for the release of serotonin. Evidence is presented suggesting that Ca2+ mobilization and C-kinase activation are synergistically involved in the physiological response of platelets to extracellular messengers, such as thrombin, collagen and platelet-activating factor.

Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters.

Tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) directly activate in vitro Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C), which normally requires unsaturated diacylglycerol. Kinetic analysis indicates that TPA can substitute for diacylglycerol and greatly increases the affinity of the enzyme for Ca2+ as well as for phospholipid. Under physiological conditions, the activation of this enzyme appears to be linked to the receptor-mediated phosphatidylinositol breakdown which may be provoked by a wide variety of extracellular messengers, eventually leading to the activation of specific cellular functions or proliferation. Using human platelets as a model system, TPA is shown to enhance the protein kinase C-specific phosphorylation associated with the release reaction in the total absence of phosphatidylinositol breakdown. Various phorbol derivatives which have been shown to be active in tumor promotion are also capable of activating this protein kinase in in vitro systems.

The evolutionary origins of hormones, neurotransmitters, and other extracellular chemical messengers: implications for mammalian biology.

IN the past, each hormonal peptide was traditionally considered a unique product of a single cell type that was localized in distribution to a limited region of the body known as an endocrine gland (Table 1). These boundaries were too limited. For some hormones, the particular type of cells producing them were also found outside those glands. In addition, cancers derived from nonendocrine tissues, as well as diverse neurons, produce hormonal peptides.1 2 3 More recently, other cell types (non-neural and nonmalignant) have also been recognized as possible sources of peptide hormones (Table 1). Because the endocrine glands of mammals did not . . .

Membrane Phospholipid Turnover, Receptor Function and Protein Phosphorylation

A variety of cellular activities are often regulated by two opposing groups of extracellular messengers. In many tissues it seems plausible that cyclic AMP (CAMP) plays a role in the inhibitory cellular processes, which may be induced by a group of hormones, neurotransmitters, prostaglandins and many other extracellular messengers. On the other hand, Ca 2+ has been proposed to play a role of crucial importance in the activation of cellular functions, although the precise mechanism of this transmembrane signaling has not yet been clarified. This chapter describes a new receptor function that is recently found in laboratory studies. This receptor function appears to be directly related to phosphatidylinositol turnover, which is induced by another group of extracellular messengers, such as acetylcholine (muscarinic), catecholamines, various peptide hormones, and many other biologically active substances, whose actions are not mediated by CAMP but are implicated in Ca 2+ gating or movement. In a manner analogous to the CAMP-dependent signal pathway, a new species of protein kinase is involved as an essential ingredient in this receptor function. This protein kinase, referred to as “protein kinase C,” is found widely in mammalian tissues, and has been purified from rat brain to near homogeneity. The enzyme absolutely requires Ca 2+ and membrane phospholipid for its activation. In addition, a small quantity of diacylglycerol derived from the receptor-linked cleavage of phosphatidylinositol dramatically increases the affinity of enzyme for Ca 2+ as well as for phospholipid, and thereby initiates the activation of enzyme at less than micromolar concentrations of Ca 2+ .

The insulin receptor.

Cells are endowed with specific cognitive molecules that function as receptors for hormones, neurotransmitters, and other intercellular messengers. The receptor molecules may be present in the plasma membrane, cytoplasm, or nucleus. When occupied by the messenger, the receptor is coupled to the cellular machinery that responds to the message-bearing molecules. For some hormones the events following attachment of the messenger to the receptor are well known. An example is the generation of cAMP after combination of glucagon with its receptor and the series of steps culminating in activation of phosphorylase. In the case of many other messengers, including insulin, the nature of these coupling steps is not known. Receptors are subject to the regulatory processes of synthesis, degradation, and conformational change; alterations in receptor properties may have significant effects on the qualitative and quantitative responses of the cell to the extracellular messenger. The insulin receptor is located in the plasma membrane, is composed of two pairs of subunits, and has a molecular weight of about 350,000. It is located in cells such as adipocytes, hepatocytes, and skeletal muscle cells as well as in cells not considered to be typical target organ cells. Insulin receptors in nonfetal cells are downregulated by exposure of the cells to high concentrations of insulin. Other factors that regulate insulin binding include muscular exercise, diet, thyroid hormones, glucocorticoids, androgens, estrogens, and cyclic nucleotides. The fetus has high concentrations of insulin receptors in several tissues. These begin to appear early in fetal life and may outnumber those found in adult tissues. Fetal insulin receptors are unusual in that they may not undergo downregulation but may experience the opposite when exposed to insulin in high concentrations. Thus the offspring of a mother with poorly controlled diabetes may be placed in double jeopardy by fetal hyperinsulinemia and augmented insulin binding by the receptors. Many disorders in children and adults are associated with changes in the properties of the insulin receptor. In general, the alterations have been measured in receptor-bearing cells that are readily accessible, such as circulating monocytes and erythrocytes. The receptors on these cells generally reflect the status of receptors on the major target organs of insulin, although exceptions are known, and conclusions drawn from studies of receptors on circulating cells must be made with caution.(ABSTRACT TRUNCATED AT 400 WORDS)

Taurine release from the pineal gland is stimulated via a β-adrenergic mechanism

Pineal gland were prelabeled in organ culture with [ 14Ctaurine, and then treated with biogenic amines. The rate of release of [ 14Ctaurine was rapidly elevated by low concentrations of norepinephrine, apparently acting through a β-adrenergic mechanism. The release was probably not due to a nonspecific change in membrane permeability, as a comparable release was not seen with [ 14Cglycine and [ 14Cα-aminoisobutyric acid. These findings suggest that the release of taurine may be related to or a reflection of the early events in adrenergic activation of the pineal gland, such as hyperpolarization of pinealocytes. The released taurine might also have an extracellular role as it has been recently shown that extracellular taurine can interact with pineal β-adrenergic receptors to stimulate melatonin production. Thus, the released taurine could possibly act as an extracellular feedback messenger.

Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol, its possible relation to phosphatidylinositol turnover.

Ca2+-activated, phospholipid-dependent protein kinase from various mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y (1979) J. Biol. Chem. 254, 3692-3695) was greatly stimulated by the addition of diacylglycerol at less than 5% (w/w) the concentration of phospholipid. This stimulation was due to an increase in the apparent affinity of enzyme for phospholipid and to a concomitant decrease in the Ka value for Ca2+ from about 1 x 10(-4) M to the micromolar range. Diacylglycerol alone showed little or no effect on enzymatic activity over a wide range of Ca2+ concentrations. This effect was greatest for diacylglycerol which contained unsaturated fatty acid at least at position 2. The active diacylglycerols so far tested included diolein, dilinolein, diarachidonin, 1-stearoyl-2-oleoyl diglyceride, and 1-stearoyl-2-linoleoyl diglyceride. In contrast, diacylglycerols containing saturated fatty acids such as dipalmitin and distearin were far less effective. Triacyl- and monoacylglycerols were totally ineffective, irrespective of the fatty acyl moieties. Cholesterol and free fatty acids were also ineffective. Based on these observations, a possible coupling is proposed between the protein kinase activation and phosphatidylinositol turnover which can be provoked by various extracellular messengers.

Regulatory peptides of the hypothalamus.

The peptides that are the principal subject of this review were originally detected in extracts of the hypothalamus as a result of their effects on the secretory functions of the anterior pituitary gland. According to the portal-vessel chemotransmitter hypothesis ( 155, 192), neurosecretory cells in the hypothalamus produce factors or hormones which reach the adenohypophysis by way of the hypothalamic-hypo­ physeal portal system. These neural factors act in concert with other blood-borne signals from the periphery to regulate the functions of the various anterior pituitary cells. To date, three peptides have been purified and characterized, and furthermore shown to have unequivocal hypophysiotropic activities by numerous investigators. These are TRF, LRF, and somatostatin (SS) whose alternative names, sequences, and principal actions on the anterior pituitary are shown in Table 1 . All three of these peptides have biological actions on the central nervous system (CNS) in addition to their effects on the anterior pituitary. Furthermore, TRF, LRF, and somatostatin are widely distributed throughout the brain, and somatostatin is found in and can influence the function of the pancreas and gastrointestinal tract as well. In general, the hypothalamic regulatory peptides (HRP) appear to act as local extracellular signals or messengers, as cells containing (producing) the HRP are found in, are adjacent to, or have restrictive vascular or possibly cerebral spinal-fluid links to cells that can respond to these peptides. Peptides with such local actions have been termed members of the paracrine rather than the endocrine system ( 152). The observations indicating that these peptides are exclusively neither hypo­ thalamic nor hypophysiotropic present problems of classification as well as nomen­ clature to neuroendocrinologists. In the interest of historical continuity, we continue to refer to these peptides as hypothalamic and maintain the original releasing­ factor terminology until such time as IUPAC-IUB-approved trivial names gain acceptance. Analyses for the purpose of categorizing the HRP as hormones, neurotransmitters, or a separate class of extracellular messengers reveal the