Attraction by repellents: an error in sensory information processing by bacterial mutants.

Normal Escherichia coli bacteria are repelled by acetate, benzoate, and indole and attracted by alpha-aminoisobutyrate. We have isolated mutants that are attracted to acetate, benzoate, and indole and may be repelled by alpha-aminoisobutyrate. These reversed-taxis mutants are defective in a central processing component: a set of methylated proteins known as MCP 1. The mechanism of reversal of taxis is discussed.

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.

cGMP: the wayward child of the cyclic nucleotide family.

An informal poll of neurobiologists indicates the following widely-held misconceptions about cGMP: (1) we know very little about it; (2) it must not be very different from cAMP; and (3) no new biological principles are likely to emerge from studying it. In fact, despite these prejudices, our understanding of the cGMP second messenger cascade has increased dramatically in the last few years. We now know that it is very different from the cAMP system in almost every particular, and the differences reveal interesting and novel solutions to the biological problem of receptor-effector coupling.

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine.

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [125I]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

Tissue distribution, cellular source, and structural analysis of rat immunoreactive uroguanylin.

Uroguanylin, a member of the guanylin peptide family, acts on guanylyl cyclase C (GC-C) to regulate intestinal and renal fluid and electrolyte transport through the second messenger, cGMP. Using an antiserum raised against synthetic rat uroguanylin, we established an RIA and identified three endogenous molecular forms in the intestine and kidney: a 15-amino acid uroguanylin, an 18-amino acid uroguanylin that is a monobasic processing product, and a 9.4-kDa prouroguanylin. Prouroguanylin is the major molecular form in these two tissues, whereas only 15-amino-acid uroguanylin is present in the urine. Rat uroguanylin is most abundant in the proximal small intestine, its content decreasing toward the colon. Uroguanylin is present immunohistochemically in the endocrine cells in the intestine and stomach, B cells in the pancreatic islets, and tubular epithelial cells in the kidney. Uroguanylin has a widespread tissue distribution and is located in cells that function in an endocrine, paracrine, and/or luminocrine (luminal secretion) fashion. Uroguanylin may have physiological functions other than the regulation of fluid and electrolyte transport in the intestine and kidney.

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain.

Cyclic AMP and cyclic GMP serve as second messengers in a variety of neural cells, modulating their metabolic and electrical activity. The cyclic GMP-stimulated cyclic nucleotide phosphodiesterase, an enzyme whose hydrolytic activity is allosterically regulated by cyclic GMP in peripheral tissues, could play an important role in the regulation of cyclic nucleotide levels in the brain. To study the presence and distribution of cyclic GMP-stimulated phosphodiesterase in the rat brain, we cloned a portion of rat liver cyclic GMP-stimulated phosphodiesterase complementary DNA by polymerase chain reaction, using degenerate phosphodiesterase-specific oligonucleotide primers. Northern blot analysis of rat tissues reveals abundant expression of cyclic GMP-stimulated phosphodiesterase messenger RNA in the brain. Northern blot analysis of brain subregions shows especially strong expression in hippocampus and cortex, modest expression in the remainder of the forebrain and in the midbrain, and little expression in cerebellum and hindbrain. In situ hybridization studies with cyclic GMP-stimulated phosphodiesterase riboprobes confirm these northern blot results, and delineate cell groups with high levels of expression. Medial habenular nucleus is intensely labeled, as is hippocampus in the vicinity of pyramidal and granule cell bodies in areas CA1, CA2, CA3, and dentate gyrus. Other elements of the limbic system also contain cyclic GMP-stimulated phosphodiesterase messenger RNA, including olfactory and entorhinal cortices, subiculum, and amygdala. Additional cortical regions show more diffuse expression of cyclic GMP-stimulated phosphodiesterase messenger RNA, as do the basal ganglia. Cerebellum, thalamus, and hypothalamus do not show appreciable specific labeling. These studies demonstrate the presence of cyclic GMP-stimulated phosphodiesterase messenger RNA in specific regions of the rat brain, and suggest that the cyclic GMP-stimulated phosphodiesterase might modulate neuronal activity by regulating intracellular cyclic AMP levels in response to changes in intracellular cyclic GMP levels.

Purification, cDNA sequence, and tissue distribution of rat uroguanylin.

Guanylin, a peptide purified from rat jejunum, is thought to regulate water and electrolyte balance in the intestine. We show here, using a combination of Northern blots, Western blots, and functional assays, that guanylin and its receptor (GCC) are not distributed in parallel within the rat intestine. To investigate the possibility that there might be a second intestinal peptide that serves as a ligand for GCC, we assayed tissue extracts for the ability to stimulate cyclic GMP synthesis in a GCC-expression cell line. Duodenal extracts display a peak of biological activity that is not present in colon and that does not comigrate with guanylin or proguanylin. The activity co-purifies with a novel peptide (TIATDECELCINVACTGC) that has high homology with uroguanylin, a peptide initially purified from human and opossum urine. A rat uroguanylin cDNA clone was found to encode a propeptide whose C-terminus corresponds to our purified peptide. Northern blots with probes generated from this clone reveal that prouroguanylin mRNA is strongly expressed in proximal small intestine, but virtually absent from colon, corroborating our biochemical measurements. Taken together, these studies demonstrate an intestinal origin for uroguanylin, and show that within the intestine its distribution is complementary to that of guanylin.

Activation of membrane guanylate cyclase by an invertebrate peptide hormone.

Peptide hormones can stimulate cyclic GMP synthesis through either of two general mechanisms: some peptides activate the cytoplasmic form of guanylate cyclase via a coupling factor called EDRF (endothelium-derived relaxation factor), while others activate the membrane form by interacting directly with an extracellular binding domain of the cyclase molecule itself. We have investigated the mechanism(s) by which crustacean hyperglycemic hormone (CHH), a neuropeptide that regulates energy metabolism in crustaceans, elevates cyclic GMP levels in lobster muscle. Phosphodiesterase inhibitors potentiate the response in intact tissue. This indicates that the primary effect of the peptide is to activate a cyclase rather than inhibit a phosphodiesterase. Methylene blue, a specific inhibitor of the EDRF pathway, does not block the actions of CHH. In addition, nitroprusside, an agent that directly activates the EDRF pathway in vertebrate animals, does not activate guanylate cyclase either in intact or homogenized lobster muscle. This indicates that the EDRF pathway, although prominent in vertebrate muscle, is not found in crustaceans and further suggests that the membrane cyclase is the most likely target of CHH. Membrane and soluble cyclases can be isolated from homogenates of lobster muscle (in a 3.5:1 ratio), and both are stimulated by Mn2+ and inhibited by Ca2+. CHH has no effect on the soluble enzyme. Coupling of CHH receptors to the particulate cyclase, however, remains intact in isolated membranes, thus providing a new model system for the study of receptor/cyclase interactions.

Purification and chemical characterization of peptide G1, an invertebrate neuropeptide that stimulates cyclic GMP metabolism.

Bioassay analysis of extracts of the major neurosecretory structures of the American lobster have revealed several different agents with stimulatory effects on the cyclic GMP metabolism of various lobster tissues. The most potent of these is a peptide extracted from the sinus gland, a neurohemal organ found in the animal's eyestalk. This molecule, called peptide G1 (for its effects on cyclic GMP metabolism), can increase the cyclic GMP content of every lobster tissue tested, sometimes by as much as 200-fold. In this article, we describe the purification and some of the chemical properties of peptide G1. Purification was accomplished by sequential anion exchange and reverse-phase HPLC. The purified peptide is a large, extremely hydrophobic molecule. Its apparent molecular mass on a reducing sodium dodecyl sulfate-containing gel is 6.4 kDa, and its calculated molecular mass (based on an amino acid analysis of the purified material) is 8.2 kDa. Amino acid analysis reveals a high proportion of leucine and valine residues. The amino terminus of the molecule is not susceptible to Edman degradation, but sequencing studies were successfully carried out on tryptic fragments. Based on the estimated size of the molecule, these studies provide approximately 60% of the total sequence. No homologies with any previously sequenced peptide were observed, but biochemical similarities to as yet unsequenced peptides found in extracts of sinus glands from other crustaceans (hyperglycemic hormone and moult-inhibiting hormone) are described.

Cyclic AMP only partially mediates the actions of serotonin at lobster neuromuscular junctions.

Serotonin (5-HT) has multiple physiological actions at lobster neuromuscular junctions, including facilitation of transmitter release from nerve terminals and an increase in the tone and excitability of muscle fibers. These physiological effects of 5-HT are accompanied by a rise in intracellular levels of cAMP. We have used combined biochemical and physiological approaches to investigate whether cAMP directly mediates the physiological actions of the hormone. Based on the following lines of evidence, we conclude that the postsynaptic increase in muscle tone occurs independently of cAMP and that while the cyclic nucleotide does play a role in the facilitation of transmitter release by 5-HT, there is also a cAMP-independent component to this facilitation. (1) Agents that mimic the action of 5-HT on cAMP levels (forskolin, IBMX, SQ20009, 8-bromo cAMP) fail to mimic the postsynaptic actions of the amine. These agents do facilitate transmitter release, although none of them has as large an effect as does 5-HT. (2) When 5-HT is removed, presynaptic facilitation decays as the sum of 2 exponentials with very different time courses. The rate of the slower process is similar to the rate of breakdown of cAMP, while the faster process and the postsynaptic response decay significantly more rapidly. (3) IBMX retards the breakdown of cAMP and simultaneously retards the decay of the slower presynaptic process, with little or no effect on the other responses. (4) IBMX and forskolin potentiate the effect of 5-HT on cAMP levels and selectively enhance the slowly decaying presynaptic component with little or no effect on the other responses.

Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA.

Guanylate cyclases play a role in both physiological and pathological secretion in the mammalian intestine. Agents that raise guanosine 3',5'-cyclic monophosphate (cGMP) levels, such as atrial natriuretic peptide (ANP), guanylin (an endogenous intestinal peptide), or Escherichia coli heat-stable enterotoxin type a (STa; a bacterial toxin), enhance electrolyte secretion and the accumulation of luminal fluid. Although secretion in all parts of intestine is sensitive to changes in cGMP metabolism, an increasing body of evidence suggests that these responses are particularly important in proximal colon. To date, three peptide-sensitive membrane-bound guanylate cyclases [types A, B, and C (GCA, GCB, and GCC, respectively)] have been cloned from mammalian tissues. GCA responds to ANP, GCB to C-type natriuretic peptide, and GCC to guanylin and STa. Expression of these receptor/cyclase genes has not previously been investigated at the cellular level in the colon. Nucleotide probes specific for GCA, GCB, GCC, and guanylin were generated by polymerase chain reaction. These probes were used to evaluate colonic cyclase and guanylin mRNA expression in the rat. GCB mRNA is not detectable in this tissue either by in situ hybridization or by Northern blot analysis. In contrast, GCA, GCC, and guanylin mRNAs are all conspicuously expressed. With the in situ hybridization technique, GCA mRNA expression is seen in cells in the lamina propria. GCC mRNA expression is seen in epithelial cells throughout colonic crypts, and also, although at a slightly lower level, in cells of the surface epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensory transduction in Escherichia coli: a requirement for methionine in sensory adaptation.

Chemotaxis of E. coli is a behavioral response to a change in the concentration of a stimulatory compound. The response is transient; thus, E. coli undergoes sensory adaptation. In this communication, we show that L-methionine is required by E. coli for adaptation to increases in the concentration of chemical attractants, but is not required for the maintenance of the adapted state. When the concentration of the attractant is lowered to its initial level, cells regain their sensitivity to the attractant. This process of deadaptation does not require methionine. We suggest that the methylation of a membrane protein, a reaction previously shown to be involved in chemotaxis [Kort, E.N., Goy, M.F., Larsen, S.H. & Adler J. (1975) Proc. Natl. Acad. Sci. USA 72, 3939-3943] underlies these phenomena.

Identification and characterization of a polypeptide from a lobster neurosecretory gland that induces cyclic GMP accumulation in lobster neuromuscular preparations.

Several observations suggest that cyclic GMP might regulate some aspect of neuromuscular physiology or metabolism in the lobster. Homarus americanus: lobster muscle is one of the richest known sources of cyclic GMP-dependent protein kinase, the preparation contains several phosphoproteins whose state of phosphorylation is affected by cyclic GMP more effectively than by cyclic AMP, and guanylate cyclase and phosphodiesterase are active in this tissue. However, no factor has yet been identified that alters lobster muscle cyclic GMP levels. We have screened extracts of neural and neurosecretory structures for the capacity to promote cyclic GMP accumulation in isolated exoskeletal muscles. Extracts of the sinus gland (a neurohemal organ found in the eyestalk) contain a factor that induces up to 100-fold increases in muscle cyclic GMP content, whereas extracts of other tissues are ineffective. This factor can also act on targets other than muscle, with hepatopancreas, testis, and neuronal tissue showing the largest responses. The sinus gland factor does not appear to affect cyclic GMP metabolism by depolarizing the preparation or by mobilizing extracellular Ca2+. The effect on cyclic GMP levels is dose-dependent and linear with time. Biological activity is destroyed by boiling and by 90% ethanol. It is also destroyed by trypsin, chymotrypsin, or pronase, which suggests that the factor is a protein or peptide. Both gel filtration chromatography and experiments using dialysis tubing with different molecular weight exclusion limits indicate that the factor has an apparent molecular weight of 5,000-12,000 daltons. A preliminary fractionation scheme, based on gel filtration, ion-exchange, and reverse-phase chromatography, gives greater than 1,300-fold purification. Our long-range goal is to purify this factor to homogeneity, compare it to other peptide hormones, and use it as a probe to evaluate the role of cyclic GMP at the neuromuscular junction.

Synthesis and vectorial export of cGMP in airway epithelium: expression of soluble and CNP-specific guanylate cyclases.

Guanosine 5'-cyclic monophosphate (cGMP) is an important modulator of fluid balance in many epithelia. We examined its metabolism in primary cultures of human airway epithelia. Sodium nitroprusside increased cGMP levels 30-fold, suggesting that the respiratory epithelium expresses a soluble guanylate cyclase; however, endogenous nitric oxide production was not detected. cGMP levels could also be increased by C-type natriuretic peptide (CNP), but not by atrial natriuretic peptide, brain natriuretic peptide, or Escherichia coli heat-stable enterotoxin, indicating expression of a CNP-specific membrane-bound guanylate cyclase. The one-half effective concentration for CNP was 40 nM and the maximal velocity was 56.7 pmol cGMP.mg protein-1.h-1. After CNP stimulation, approximately 60% of the total synthesized cGMP was preferentially exported from the polarized epithelial cells across the basolateral membrane by a probenecid-sensitive process. Isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) export revealed a similar export pattern and probenecid sensitivity, although a lower efficiency of export (27% of total cAMP was exported). Consistent with previous reports, export of neither cyclic nucleotide was saturable at the concentrations tested. We conclude that the respiratory epithelium expresses a soluble guanylate cyclase, a CNP-specific receptor, and a novel vectorial cyclic nucleotide export mechanism.

Uroguanylin is expressed by enterochromaffin cells in the rat gastrointestinal tract.

BACKGROUND & AIMS: Guanylin and uroguanylin are recently discovered intestinal peptides. Identifying the type of cell that synthesizes and secretes each peptide is an important step toward defining their physiological functions. However, the site of uroguanylin expression has not been identified, and the site of guanylin expression remains controversial (some studies implicate goblet cells, whereas others implicate enterochromaffin cells). The aim of the present study was to identify cellular sites of uroguanylin expression in the rat gastrointestinal tract and resolve the guanylin localization controversy. METHODS: Polyclonal antibodies against two independent regions of the uroguanylin prohormone were raised and used to evaluate prouroguanylin expression by Western blotting and immunohistochemistry. In parallel, uroguanylin mRNA was localized by in situ hybridization. RESULTS: Uroguanylin propeptide expression is high in proximal small intestine, low in stomach and distal small intestine, and almost undetectable in large intestine. Uroguanylin-expressing cells are identified as a subpopulation of enterochromaffin cells. CONCLUSIONS: Previous results showing guanylin expression in enterochromaffin cells appear to be a consequence of antibody cross-reactivity for epitopes conserved between proguanylin and prouroguanylin. Expression of uroguanylin in enterochromaffin cells is consistent with the hypothesis that uroguanylin is secreted both apically (into the lumen) and basolaterally (into the circulation).

Peptide F1, an N-terminally extended analog of FMRFamide, enhances contractile activity in multiple target tissues in lobster.

The physiological actions of lobster peptide F1 (TNRNFLRFamide) have been examined on three different lobster nerve-muscle preparations (exoskeletal, cardiac and visceral). The peptide, which is found at high concentrations in a lobster neurosecretory gland, causes a long-lasting enhancement of contractility in each target tissue. On exoskeletal nerve-muscle preparations, peptide F1 has the following actions: (1) it potentiates transmitter release from nerve terminals innervating exoskeletal muscle, leading to an increase in both spontaneous and nerve-evoked release of transmitter; (2) it acts directly on the muscle, in the absence of nerve activity, to induce tonic contractions; and (3) it shows a potent desensitization that does not reverse with prolonged washing of the tissue. On each of the types of muscle examined, peptide F1 is active at nanomolar concentrations and is 3-4 orders of magnitude more potent than FMRFamide. These findings suggest that peptide F1 is a neurohormone with widespread myogenic actions throughout lobster peripheral tissues. The molecular mechanism(s) by which the peptide acts are not yet known, but do not appear to involve cyclic AMP or cyclic GMP.

Sensory transduction in Escherichia coli: role of a protein methylation reaction in sensory adaptation.

The behavioral response of Escherichia coli to the addition of a stimulatory compound is transient; thus the organism undergoes sensory adaptation. When the compound is removed, E. coli undergoes the inverse process, called deadaptation, and very rapidly regains its sensitivity to the stimulus. In this communication we demonstrate that the previously reported methylation of several cytoplasmic membrane proteins is correlated with, and very likely controls, the state of adaptation of the cell. In the absence of an added stimulus these proteins are methylated to a basal level. When the bacteria are stimulated by the addition of an attractant, the extent of methylation increases over a period of several minutes to a new level, which is maintained as long as the attractant is present. The magnitude of the increase in methylation is a function of the size of the stimulus and is directly proportional to the duration of the behavioral response. Upon removal of the attractant the level of methylation very rapidly falls to the basal value. Previously we have shown that adaptation requires methionine, but maintenance of the adapted state and de-adaptation do not [Springer, M. S., Goy, M. F. & Adler, J. (1975) Proc. Natl. Acad. Sci. USA 74, 183-187]; here we demonstrate that methylation requires methionine but maintenance of an attractant-induced level of methylation and the demethylation that occurs following removal of the attractant do not. These results strongly indicate a role for protein methylation in sensory adaptation.

Sensory transduction in Escherichia coli: two complementary pathways of information processing that involve methylated proteins.

The properties of two classes of behavioral mutants of Escherichia coli (called tsr and tar) are described. The mutations in these strains define two complementary pathways of information flow in bacterial chemotaxis: behavioral responses to one set of stimuli are defective in tsr mutants, while responses to a complementary set of stimuli are defective in tar mutants. A double mutant containing both genetic lesions is defective in responses to all stimuli tested. The behavioral defects are shown to correlate with alterations in the properties of a methylation reaction involved in chemotaxis. Two independent sets of methyl-accepting proteins are demonstrated in the wild type, each set functioning in one of the two pathways mentioned above. Methylation of one set of proteins is defective in tsr mutants, while methylation of the complementary set is defective in tar mutants. The double mutant shows no methylation of either set. The relationship between the genetic loci (tsr and tar) and the methyl-accepting proteins is discussed.

Protein methylation in behavioural control mechanisms and in signal transduction.

In both prokaryotes and eukaryotes methyl groups can be added to and removed from the carboxyl groups of proteins. Recent work has revealed that these reactions have a role in several behavioural phenomena. The nature of this role has been uncovered in one case--that of bacterial chemotaxis.

Failure of sensory adaptation in bacterial mutants that are defective in a protein methylation reaction.

Chemotactic bacteria, such as E. coli, detect changes in the chemical composition of the environment. Addition of an attractant or repellent leads to an immediate response, characterized by a change in the swimming behavior of the cells--a process known as sensory excitation. However, the response gradually disappears with time, despite the continued presence of the chemical--a process known as sensory adaptation. We report here the behavior of a class of nonchemotactic mutants (cheX) that can carry out sensory excitation but are defective in the process of sensory adaptation. These mutants are also defective in the ability to carry out a protein methylation reaction which has previously been implicated in the adaptation process (Goy, Springer and Adler, 1977). The results presented here establish a firm relationship between the methylation reaction and sensory adaptation.