Ca-modulated ONE-GC odorant signal transduction

In a subset of olfactory epithelium the odorant receptor guanylate cyclase, ONE-GC, is a central transduction component of the cyclic GMP signaling pathway. The odorant binds to the extracellular domain and activates its intracellular catalytic domain to generate the odorant second messenger, cyclic GMP. The present study demonstrates that it is a two-step, Ca²⁺-independent and Ca²⁺-dependent, sequential process. In step one, the odorant, uroguanylin, binds ONE-GC and primes it for stimulation. In step two, Ca²⁺-bound neurocalcin δ binds to the defined intracellular domain and saturates ONE-GC activity. A prototype model is proposed that depicts this signal transduction process.     

ONE-GC membrane guanylate cyclase, a trimodal odorant signal transducer

The Ca²⁺-modulated ONE-GC membrane guanylate cyclase is a central component of the cyclic GMP signaling in odorant transduction. It is a single transmembrane spanning modular protein. Its intracellular region contains Ca²⁺ sensor recognition domains linked to GCAP1 and to neurocalcin δ, and a catalytic module. These domains sense increments in free Ca²⁺ and stimulate the catalytic module. The present study makes three significant mechanistic advancements. First, to date no ligand for the extracellular (ext) domain is known, for this reason ONE-GC has been deemed as an orphan receptor. The present study identifies its ligand. Uroguanylin stimulates ONE-GC through its ext domain. Second, so far no ligand is known that directly stimulates the catalytic module of any membrane guanylate cyclase. The presented evidence shows that in the presence of the semimicromolar range of free Ca²⁺, neurocalcin binds to the catalytic module and stimulates ONE-GC. Thus, ONE-GC has trimodal regulation, two occurring intracellularly and one extracellularly. Third, guanylin, a urine odorant, does not directly stimulate ONE-GC. This challenges the proposed hypothesis that the guanylin odorant signal occurs via ONE-GC [T. Leinders-Zufall, R.E. Cockerham, S. Michalakis, M. Biel, D.L. Garbers, R.R. Reed, F. Zufall, S.D. Munger, Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium, Proc. Natl. Acad. Sci. USA. 104 (2007) 14507-14512].     

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca²⁺ sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca²⁺-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca²⁺ myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca²⁺ myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca²⁺-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca²⁺-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.     

Interaction of atrial natriuretic factor and endothelin-1 signals through receptor guanylate cyclase in pulmonary artery endothelial cells

The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [¹²⁵I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC₅₀ of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC₅₀ of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.     

S100B-modulated Ca2+-dependent ROS-GC1 transduction machinery in the gustatory epithelium: a new mechanism in gustatory transduction

Gustatory transduction is a biochemical process by which the gustatory signal generates the electric signal. The microvilli of the taste cells in the gustatory epithelium are the sites of gustatory transduction. This study documents the biochemical, molecular, and functional identity of the Ca2+-modulated membrane guanylate cyclase transduction machinery in the bovine gustatory epithelium. The machinery is a two-component system: the Ca2+-sensor protein, S100B; and the transducer, ROS-GC1. S100B senses increments in free Ca2+, undergoes conformational change, binds to the domain amino acids (aa) Gly962-Asn981 and via the transduction domain aa Ile1030-Gln1041 activates ROS-GC1, generating the second messenger, cyclic GMP. In a recent study, operational presence of this machinery has been demonstrated in the photoreceptor bipolar synapse [Duda et al., EMBO J. 21 (2002) 2547]. Thus, the machinery has a broader role in sensory perceptions, vision in the retinal neurons and gustation in the tongue. The entry of the ROS-GC transduction machinery defines the beginning of a new paradigm of Ca2+ signaling in the tongue.     

Photoreceptor Guanylate Cyclases: A Review

Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca²⁺ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca²⁺ signals. The low Ca²⁺ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca²⁺ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca²⁺ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in on-bipolar cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Two membrane juxtaposed signaling modules in ANF-RGC are interlocked

Atrial natriuretic factor (ANF) receptor guanylate cyclase ANF-RGC is a single transmembrane spanning modular protein. Juxtaposed to each side of the transmembrane module is a Cys423-Cys432 disulfide ANF signaling module motif and the ATP-regulated transduction module (ARM) motif. The signaling module motif is conserved in nearly all membrane guanylate cyclases and is believed to be critical in the signaling activities of all membrane guanylate cyclases. The present study with the model system of the olfactory membrane guanylate cyclase shows that this concept is not valid. Furthermore, the study shows that in ANF-GC the signaling motif works through the ARM domain. A new signaling model is proposed where in its natural state the disulfide structural motif represses the ARM domain activity, which, in turn, represses the catalytic module activity of ANF-RGC. ANF signaling relieves the disulfide structural motif restraint on the ARM inhibition and stimulates the catalytic module of the cyclase.     

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTPγS), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t_1.2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca²⁺ ions inhibited the enzyme (half-maximal effect at 0.3 μM), whereas InsP₃ had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells. © 1996 Wiley-Liss, Inc.     

Interleukin-1α (IL-1α) production by alveolar macrophages in patients with acute lung diseases: the influence of zinc supplementation

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critcal role in visual transduction. But regulation of the cyclase by some of these factors — phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn) — is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.This work was supported by the grants from the National Institutes of Health (EY07158, EY 05230, EY 10828, NS 23744) and the equipment grant from Pennsylvania Lions Eye Research Foundation.     

ATP signaling site in the ARM domain of atrial natriuretic factor receptor guanylate cyclase

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single transmembrane spanning modular protein. It binds ANF to its extracellular module and activates its intracellular catalytic module located at its carboxyl end. This results in the accelerated production of cyclic GMP, which acts as a critical second messenger in decreasing blood pressure. Two mechanistic models have been proposed for the ANF signaling of ANF-RGC. One is ATP-dependent and the other ATP-independent. In the former, ATP works through the ARM (ATP-regulated transduction module) of ANF-RGC. This model has recently been challenged [Antos et al. (2005) J Biol Chem 280:26928-26932] in support of the ATP-independent model. The present in-depth study analyzes the major principles of this challenge and concludes that the challenge lacks merit. The study then moves on to dissect the ATP mechanism of ANF signaling of ANF-RGC. It shows that the ATP photoaffinity probe, [gamma(32)P]-8-azido-ATP, reacts with Cys(634) residue in the ATP-binding pocket of ARM, and also signals the ANF-dependent activation of ANF-RGC. The target site of the 8-azido (nitrene) group is between the Cys(634) and Val(635) bond of the ATP-binding pocket. Thus, the study experimentally validates the ARM model-predicted role of Val(635) in the folding pattern of the ATP-binding pocket. And, it also identifies another residue Cys(634) that along with eight already identified residues is a part of the fold around the adenine ring of the ATP pocket. This information establishes the direct role of ATP in ANF signal transduction model of ANF-RGC, and provides a significant advancement on the mechanism by which the ATP-dependent transduction model operates.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

ATP modulation of the ligand binding and signal transduction activities of the type C natriuretic peptide receptor guanylate cyclase

The type C natriuretic peptide (CNP)-activated guanylate cyclase (CNP-RGC) is a single-chain transmembrane-spanning protein, containing both CNP binding and catalytic cyclase activities. Upon binding CNP to the extracellular receptor domain, the cytosolic catalytic domain of CNP-RGC is activated, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening signal transduction step which is regulated by ATP binding to the cyclase. This bridges the events of ligand binding and cyclase activation. A defined sequence motif (Gly⁴⁹⁹-Xa-Xa-Xa-Gly⁵⁰³), termed ATP regulatory module (ARM), is critical for this step. The present study shows that ATP not only amplifies the signal transduction step, it also concomitantly reduces the ligand binding activity of CNP-RGC. Reduction in the ligand binding activity is a consequence of the transformation of the high affinity receptor-form to the low affinity receptor-form. A single ARM residue Gly⁴⁹⁹ is critical in the mediation of both ATP effects, signal transduction and ligand binding activity of the receptor. Thus, this residue represents an ATP bimodal switch to turn the CNP signal on and off.     

WTAPELL motif of the photoreceptor ROS-GC1: A general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca²⁺]_i signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca²⁺ transmitter switch (Ca²⁺TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is ⁶⁵⁷WTAPELL⁶⁶³. The two Ca²⁺ signaling GCAP pathways converge in Ca²⁺TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca²⁺-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

Calmodulin selectively modulates the guanylate cyclase activity by repressing the lipid phase separation temperature in the inner half of the bilayer of rat brain synaptosomal plasma membranes

The association of [¹²⁵I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 M Ca²⁺, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 M Ca²⁺. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)-1]^–1. Arrhenius-type plots of [(r_o/r)-1]^–1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 M Ca²⁺. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F^–) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.     

Formation of pyrophosphate by soluble guanylate cyclase from rat lung.

The guanylate cyclase reaction was studied to determine the identity of the product(s) formed other than guanosine-3′,5′-monophosphate (cyclic GMP). Partially purified guanylate cyclase preparations from rat lung catalyzed the formation of nearly equal amounts of PP₁ and of cyclic GMP from GTP. Column chromatography of the enzyme preparation on DEAE-Sephadex or Bio-Gel A-5m failed to separate the enzyme(s) involved in formation of cyclic GMP and of PP₁. Nucleotide inhibitors of cyclic GMP formation also inhibited PP₁ formation, and Ca²⁺, a stimulant of cyclic GMP formation in the presence of Mn²⁺, also stimulated PP₁ formation. Detectable PP₁ formation was not observed when ATP was present instead of GTP.The results show that guanylate cyclase, in vitro, catalyzes the formation of pyrophosphate from GTP concomitant with the synthesis of cyclic GMP.     

Nitric oxide inhibits neutrophil β2 integrin function by inhibiting membrane-associated cyclic GMP synthesis

The aim of this investigation was to identify the mechanism by which nitric oxide inhibits neutrophil β₂ integrin dependent adherence. Isolated rat neutrophils from blood and peritoneal exudates were exposed for 2 min to nitric oxide generated by diethylamine-NO at rates between 1.6 and 138 nmol/min. Exposure to nitric oxide at rates less than 14 nmol/min had no effect on adherence. Exposure to 14 to 56 nmol nitric oxide/min inhibited β₂ integrin dependent adherence to endothelial cells, nylon columns, and fibrinogen-coated plates, but higher concentrations had no significant effect on adherence. Adherence by β₂ integrins could be restored by incubating cells with dithioerythritol, phorbol 12-myristate 13-acetate, or 8-bromo cyclic GMP. Elevations in cellular cyclic GMP concentration were associated with adherence, but this did not occur after cells were exposed to concentrations of nitric oxide that inhibited β₂ integrin-dependent adherence. Elevations in cyclic GMP did occur after cells were incubated with dithioerythritol or phorbol 12-myristate 13-acetate. Concentrations of nitric oxide that inhibited β₂ integrin-dependent adherence also inhibited catalytic activity of membrane associated guanylate cyclase and binding of atrial natriuretic peptide, but were insufficient to activate cytosolic guanylate cyclase. Nitric oxide did not inhibit neutrophil oxidative burst or degranulation, nor effect β₂ integrin expression or adherence that did not depend on β₂ integrins, nor cause oxidative stress identified in terms of cellular glutathione concentration or protein nitrotyrosine. The results indicate that nitric oxide inhibited β₂ integrins in a concentration-dependent fashion by inhibiting cell-surface transduction of signals linked to the activity of membrane-bound guanylate cyclase. The inhibitory effect could be overcome by providing cells with cyclic GMP exogenously or by stimulating cytosolic guanylate cyclase. J. Cell. Physiol. 172:12–24, 1997. © 1997 Wiley-Liss, Inc.     

Activation of Brain Guanylate Cyclase by Phospholipase A2

Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane-bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.     

Lack of function of histamine H1 and muscarinic acetylcholine receptors of mouse neuroblastoma cells grown in serum-free medium

Summary Mouse neuroblastoma cells (Clone N1E-115) were grown in serum-free (defined) medium, defined medium supplemented with serum, and control medium to determine whether serum-free medium could substitute for serum-containing medium in our studies of the histamine H₁ and muscarinic acetylcholine receptors of these cells. The function of these receptors as determined by measurement of receptor-mediated cyclic [³H]GMP formation was absent in cells grown in serum-free medium and increased as the percentage of serum was increased in the defined medium, but never attained the levels found with control cells. Muscarinic receptor number for cells grown in defined medium was 60% above that found for control cells with no change in the affinity of the receptor for the radioligand (−)[³H]quinuclidinyl benzilate. Guanylate cyclase and acetylcholinesterase activities for cells grown in defined medium were 23 and 66% of those found in control cells, respectively. This marked reduction of guanylate cyclase activity in large part explains the lack of function of these receptors. Supported by Mayo Foundation and U.S. Public Health Service Grants MH27692, DA1490, and AA4443.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.