Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer.

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.

The protein kinase domain of the ANP receptor is required for signaling.

A plasma membrane form of guanylate cyclase is a cell surface receptor for atrial natriuretic peptide (ANP). In response to ANP binding, the receptor-enzyme produces increased amounts of the second messenger, guanosine 3',5'-monophosphate. Maximal activation of the cyclase requires the presence of adenosine 5'-triphosphate (ATP) or nonhydrolyzable ATP analogs. The intracellular region of the receptor contains at least two domains with homology to other proteins, one possessing sequence similarity to protein kinase catalytic domains, the other to regions of unknown function in a cytoplasmic form of guanylate cyclase and in adenylate cyclase. It is now shown that the protein kinase-like domain functions as a regulatory element and that the second domain possesses catalytic activity. When the kinase-like domain was removed by deletion mutagenesis, the resulting ANP receptor retained guanylate cyclase activity, but this activity was independent of ANP and its stimulation by ATP was markedly reduced. A model for signal transduction is suggested in which binding of ANP to the extracellular domain of its receptor initiates a conformational change in the protein kinase-like domain, resulting in derepression of guanylate cyclase activity.

Effects of extracellular egg factors on sperm guanylate cyclase.

Extracellular factors from the sea urchin egg induce a change in the electrophoretic mobility of an abundant sperm membrane phosphoprotein. The modified protein was identified as guanylate cyclase. The mobility shift of the cyclase was shown to be associated with a decrease in its enzymatic activity.

Receptor guanylyl cyclases.

Three different guanylyl cyclase cell receptors are known, but others will likely be discovered within the next few years. The general function of these receptors appear to relate to the regulation of fluid volume or fluid movement. New receptors, or possibly the currently known receptors, therefore, may be discovered in areas of the body where fluid volume regulation is important. Such fluids whose volume or composition might be regulated by guanylyl cyclase receptors include synovial fluid, uterine/oviductal luminal fluid, follicular fluid, aqueous humor, cerebral spinal fluid, seminiferous tubule luminal fluid, epididymal luminal fluid, seminal plasma, and airway luminal fluid. The function of the heterodimeric forms of guanylyl cyclase appear to relate to a primary regulation of nitric oxide (or similar molecules) concentrations, which are in turn regulated by a Ca2+/calmodulin-sensitive nitric oxide synthase.

Disruption of the guanylyl cyclase-C gene leads to a paradoxical phenotype of viable but heat-stable enterotoxin-resistant mice.

Heat-stable enterotoxins (STa), which cause an acute secretory diarrhea, have been suggested to mediate their actions through the guanylyl cyclase-C (GC-C) receptor. The GC-C gene was disrupted by insertion of neo into exon 1 and subsequent homologous recombination. GC-C null mice contained no detectable GC-C protein. Intestine mucosal guanylyl cyclase activity was approximately 16-fold higher in wild-type mice than in the GC-C null mice, and STa-stimulable guanylyl cyclase activity was absent in the null animals. Thus, GC-C is the major cyclase activity present in the intestine, and also completely accounts for the STa-induced elevations of cGMP. Gavage with STa resulted in marked fluid accumulation within the intestine of wild-type and heterozygous suckling mice, but GC-C null animals were resistant. In addition, infection with enterotoxigenic bacteria that produce STa led to diarrhea and death in wild-type and heterozygous mice, while the null mice were protected. Cholera toxin, in contrast, continued to cause diarrhea in GC-C null mice, demonstrating that the cAMP signaling pathway remained intact. Markedly different diets (high carbohydrate, fat, or protein) or the inclusion of high salt (K+, Na+) in the drinking water or diet also did not severely affect the null animals. Given that GC-C is a major intestinal receptor in all mammals, the pressure to retain a functional GC-C in the face of diarrhea-inflicted mortality remains unexplained. Therefore, GC-C likely provides a protective effect against stressors not yet tested, possibly pathogens other than noninvasive enterotoxigenic bacteria.

Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-kappaB activation and alleviates myocardial ischemia/reperfusion injury.

Acute myocardial infarction (AMI) remains the leading cause of death in developed countries. Although reperfusion of coronary arteries reduces mortality, it is associated with tissue injury. Endothelial P-selectin-mediated infiltration of neutrophils plays a key role in reperfusion injury. However, the mechanism of the P-selectin induction is not known. Here we show that infarct size after ischemia/reperfusion was significantly smaller in mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. The decrease was accompanied by decreases in neutrophil infiltration in coronary endothelial P-selectin expression. Pretreatment with HS-142-1, a GC-A antagonist, also decreased infarct size and P-selectin induction in wild-type mice. In cultured endothelial cells, activation of GC-A augmented H2O2-induced P-selectin expression. Furthermore, ischemia/reperfusion-induced activation of NF-kappaB, a transcription factor that is known to promote P-selectin expression, is suppressed in GC-A-deficient mice. These results suggest that inhibition of GC-A alleviates ischemia/reperfusion injury through suppression of NF-kappaB-mediated P-selectin induction. This novel, GC-A-mediated mechanism of ischemia/reperfusion injury may provide the basis for applying GC-A blockade in the clinical treatment of reperfusion injury.

Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior.

One of two orphan photoreceptor guanylyl cyclases that are highly conserved from fish to mammals, GC-E (or retGC1) was eliminated by gene disruption. Expression of the second retinal cyclase (GC-F) as well as the numbers and morphology of rods remained unchanged in GC-E null mice. However, rods isolated from such mice, despite having a normal dark current, recovered from a light flash markedly faster. Unexpectedly, the a- and b-waves of electroretinograms (ERG) from dark-adapted null mice were suppressed markedly. Cones, initially present in normal numbers in the retina, disappeared by 5 weeks, based on ERG and histology. Thus, the GC-E-deficient mouse defines a model for cone dystrophy, but it also demonstrates that morphologically normal rods display paradoxical behavior in their responses to light.

Demonstration of 5'-methylthioadenosine phosphorylase activity in various rat tissues. Some properties of the enzyme from rat lung.

An enzyme (5'-methylthioadenosine phosphorylase) that catalyzes the phosphorolytic cleavage of 5'-methylthioadenosine to 5-methylthioadenosine to 5-methylthioribose-1-phosphate and adenine was found in various rat tissues. Liver and lung had the highest enzyme activities and heart the lowest, most of the activity (greater than 90%) was recovered in soluble tissue fractions. The enzyme from rat lung was purified about 30-fold by pH treatment (NH4)2SO4 fractionation, and gel filtration. The enzyme did not require an added metal-ion for activity, and was not inhibited by EDTA. Many compounds were tested for their inhibitory effects; of these, ribose 1-phosphate, 2-deoxyribose 1-phosphate, fructose 1-phosphate, adenine and guanine were shown to inhibit. Kinetic patterns on reciprocal plots were linear as a function of the concentration of either 5'-methylthioadenosine or phosphate. More detailed kinetic studies suggested that the rat lung 5'-methylioadenosine phosphorylase catalyzes an equilibrium-ordered reaction, and that 5'-methylthioadenosine is the first substrate to bind and 5-methylthioribose-1-phosphate is the first product to be released.

Regulation of the phosphorylation state and function of the epidermal growth factor receptor by vitamin K-3.

Vitamin K-3 or 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced the binding of epidermal growth factor (EGF) to its receptor by more than 90% in human foreskin fibroblasts. After the equilibration of fibroblasts with [32P]orthophosphate, vitamin K-3 or TPA markedly increased the amount of 32P found in the receptor; the increase was principally due to serine and threonine phosphorylation. By the use of two-dimensional tryptic phosphopeptide mapping, using a synthetic phosphopeptide as a standard, threonine-654 was identified as one of the residues whose phosphorylation state was elevated by vitamin K-3 or TPA. Because of the large amounts of EGF receptor present on A431 human carcinoma cells, these cells were used to study further the relationship between the phosphorylation state of threonine-654, the tyrosine phosphorylation state of the receptor, and the receptor's protein tyrosine kinase activity toward exogenous substrates. Vitamin K-3 and TPA both increased the amount of phosphate on threonine-654 in A431 cells. However, whereas receptor from TPA-treated cells lacked phosphotyrosine, vitamin K-3-treated cells contained receptor with markedly elevated levels of phosphotyrosine. The addition of vitamin K-3, TPA or EGF to intact A431 cells followed by homogenization of the cells and the assay of EGF receptor protein tyrosine kinase activity by the use of a synthetic peptide substrate resulted in marked decreases in apparent receptor kinase activity. Therefore, assuming that the activity measured in the peptide assay reflects the protein tyrosine kinase activity of the receptor in the intact cell, the activity of the EGF receptor kinase cannot be deduced from the amount of phosphotyrosine associated with the receptor.

The incorporation of a purified, membrane-bound form of guanylate cyclase into phospholipid vesicles and erythrocytes.

The purified membrane-bound form of guanylate cyclase was incorporated into artificial unilamellar phospholipid vesicles. The rate and extent of enzyme incorporation into the vesicles was dependent upon the phospholipid concentration and the time period of incubation. The enzyme was incorporated at a significantly faster rate after removal of carbohydrate with endoglycosidase H. The incorporation of the enzyme led to a 10-fold decrease in the apparent maximal velocity and a 2-fold increase in the apparent Michaelis constant for MnGTP. Extraction of liposomes containing guanylate cyclase with 0.2% Lubrol PX resulted in the recovery of 85% of the original amount of added activity, suggesting that the decrease in maximal velocity was not due to enzyme denaturation. Phosphatidylcholine liposomes differentially effected the activity of the membrane-form of guanylate cyclase, dependent on the nature of the fatty acid present on the phospholipid. Specific activities ranged between 458 nmol/min per mg and 2.6 mumol/min per mg, dependent upon the fatty acids present. Liposomes containing the membrane-bound form of guanylate cyclase were subsequently fused with erythrocytes using poly(ethylene glycol) 4000 in attempts to introduce the enzyme into intact cells. The enzyme was successfully introduced into the erythrocytes; greater than 90% of the enzyme activity was subsequently shown to be associated with erythrocyte membranes. Cyclic GMP concentrations of erythrocytes increased from essentially nondetectable to 4 pmol/10(9) cells after introduction of the enzyme. These results demonstrate that guanylate cyclase can be incorporated into liposomes in an active state and that such liposomes can be used to introduce the enzyme into cells where it can subsequently function to generate cyclic GMP.

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.

The expanding family of guanylyl cyclases.

The guanylyl cyclase receptor family contains members that exist in both the particulate and soluble fractions of cell homogenates. Soluble forms of the enzyme recognize EDRF (nitric oxide) or similar molecules, while diversity within the extracellular domain of the plasma membrane forms has resulted in a series of guanylyl cyclases that are specifically activated by different ligands. David Garbers and colleagues describe the recent cloning of some of these molecules.

Guanylyl cyclase-linked receptors.

The guanylyl cyclase receptor family contains members that exist in both the particulate and soluble fractions of cell homogenates. Based on cloning studies, proteins with guanylyl cyclase activity contain a single transmembrane domain, or exist as heterodimers with no apparent transmembrane domains. The members containing the single transmembrane domain appear to act as cell surface receptors for peptides such as natriuretic peptides and bacterial heat-stable enterotoxins, while the heterodimeric forms are activated by nitric oxide. The concentrations of the intracellular messenger, cyclic GMP, then, are regulated by multiple primary signaling molecules, all of which appear to bind directly to the guanylyl cyclase enzyme.

Cyclic GMP and the second messenger hypothesis.

A plasma membrane form of guanylate cyclase appears to contain a single transmembrane domain that divides the protein into a highly conserved intracellular domain and a variable extracellular domain. Various extracellular peptides can bind directly to guanylate cyclase to increase the production of the second messenger, cyclic GMP.

Guanylyl cyclases: approaching year thirty.

Since its discovery in 1963, cyclic GMP (cGMP) has been shown to be a ubiquitous second messenger. The enzymes that catalyze the formation of cGMP from GTP, guanylyl cyclases, exist in soluble and particulate isoforms. An explosion in the number of known isoforms, gene disruption, identification of new inhibitors and activators and finally the resolution of the structure of adenylyl cyclases have all provided important clues about the structure and function of guanylyl cyclases. This article gives a brief review of the recent developments in the field of guanylyl cyclase research.

Genetic models reveal that brain natriuretic peptide can signal through different tissue-specific receptor-mediated pathways.

Brain natriuretic peptide (BNP), a hormone produced primarily by the cardiac ventricle, is thought to be involved in a variety of homeostatic processes through its cognate receptor, guanylyl cyclase A (GC-A). We previously created transgenic mice overexpressing BNP under the control of the liver-specific human serum amyloid P component promoter (BNP-transgenic mice) and demonstrated that they exhibit reduced blood pressure and cardiac weight accompanied by an elevation of plasma cGMP concentrations and marked skeletal overgrowth through the activation of endochondral ossification. To address whether BNP exerts its biological effects solely through GC-A, we produced BNP-transgenic mice lacking GC-A (BNP-Tg/GC-A-/- mice) and examined their cardiovascular and skeletal phenotypes. The GC-A-/- mice are hypertensive with cardiac hypertrophyrelative to wild-type littermates, which is not alleviated by overexpression of BNP in BNP-Tg/GC-A-/- mice. The BNP-Tg/GC-A-/- mice, however, continue to exhibit marked longitudinal growth of vertebrae and long bones comparably to BNP-Tg mice. This study provides genetic evidence that BNP reduces blood pressure and cardiac weight through GC-A, whereas it dramatically alters endochondral ossification in the absence of this receptor. Therefore, the BNP-Tg/GC-A-/- mice provide the first experimental model demonstrating that this natriuretic peptide can signal in a tissue-specific manner through a receptor other than GC-A.

New insights on the functions of the guanylyl cyclase receptors.

The discovery of at least 29 genes encoding putative guanylyl cyclases in Caenorhabditis elegans has raised the question as to whether there are numerous receptors yet to be discovered in the mammal. The nematode, however, not only seems ideal to study guanylyl cyclase receptor localization and function, given the large variety of isoforms, but also leads to possible identification of ligands for orphan guanylyl cyclases by the use of genetic and behavioral assays. A recent powerful approach to describe the function of different guanylyl cyclase isoforms in mammals has been the disruption of the corresponding genes in the mouse. A salt resistant elevation of blood pressure, which corresponds to the phenotype of 50% of all human patients with essential hypertension, is observed in mice lacking the GC-A-receptor. Mice missing the GC-C receptor have been shown to be resistant to STa, an E. coli heat-stable enterotoxin, which is largely responsible for travellers diarrhea in adults and mortality due to diarrhea in infants.

Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins.

Two peptides associated with the eggs of the sea urchin, Lytechinus pictus, which stimulate L. pictus but not Arbacia punctulata sperm respiration rates, were purified and their amino acid sequences determined. The peptides (Gly-Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln and Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln) were found to be structurally similar to the peptide, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly). Chemical synthesis of the two peptides confirmed their ability to activate sperm respiration. The peptides had equivalent biological activity with half-maximal stimulation of respiration rates and of cyclic nucleotide concentrations occurring at 60 pM and 700 pM, respectively. Addition of the peptides to intact spermatozoa resulted in the rapid appearance of a newly-stained protein on Na X dodecyl X SOl polyacrylamide gels (Mr = 140,000); one-half maximal formation of the Mr 140,000 protein occurred at about 20-100 nM peptide.

Tyrosine-specific protein kinases of normal tissues.

Tyrosine-specific protein kinases from normal tissue have been studied using synthetic peptides as substrate. Spleen had much higher activity of the enzyme in the particulate fraction than any other normal tissue (except purified T lymphocytes). The tyrosine protein kinase from the particulate fraction of rat spleen was partially purified and characterized. The kinase could phosphorylate src-related as well as unrelated peptides and casein at tyrosine residues. The enzyme in the membrane seemed to have somewhat different substrate specificity than the solubilized, partially purified enzyme. Serum containing antibody to pp60v-src did not precipitate the kinase; however, the protein kinase could phosphorylate the heavy chain of IgG from TBR serum (but not from normal serum). The possible relationship of the tyrosine-specific protein kinase of spleen with pp60c-src and other tyrosine-specific protein kinases is discussed.