Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats

The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

Pharmacophore based approach to design inhibitors in Crustaceans: An insight into the molt inhibition response to the receptor guanylyl cyclase.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms

Physiological evidence indicates that the supraoptic nucleus (SON) is an important region for integrating information related to homeostasis of body fluids. Located bilaterally to the optic chiasm, this nucleus is composed of magnocellular neurosecretory cells (MNCs) responsible for the synthesis and release of vasopressin and oxytocin to the neurohypophysis. At the cellular level, the control of vasopressin and oxytocin release is directly linked to the firing frequency of MNCs. In general, we can say that the excitability of these cells can be controlled via two distinct mechanisms: 1) the intrinsic membrane properties of the MNCs themselves and 2) synaptic input from circumventricular organs that contain osmosensitive neurons. It has also been demonstrated that MNCs are sensitive to osmotic stimuli in the physiological range. Therefore, the study of their intrinsic membrane properties became imperative to explain the osmosensitivity of MNCs. In addition to this, the discovery that several neurotransmitters and neuropeptides can modulate their electrical activity greatly increased our knowledge about the role played by the MNCs in fluid homeostasis. In particular, nitric oxide (NO) may be an important player in fluid balance homeostasis, because it has been demonstrated that the enzyme responsible for its production has an increased activity following a hypertonic stimulation of the system. At the cellular level, NO has been shown to change the electrical excitability of MNCs. Therefore, in this review, we focus on some important points concerning nitrergic modulation of the neuroendocrine system, particularly the effects of NO on the SON.

Angiotensin-II down-regulates cardiac natriuretic peptide receptor-A mediated anti-hypertrophic signaling in experimental rat hearts.

Atrial natriuretic peptide (ANP) exerts anti-hypertrophic effects in the heart via natriuretic peptide receptor-A (NPR-A). However, ANP mediated anti-hypertrophic activity is decreased in the cardiomyopathic conditions. In the present investigation the in vivo effects of angiotensin II (Ang II), a hypertrophic agonist have been studied on the ventricular expression level of NPR-A in Wistar rat hearts. NPR-A expression at the protein and mRNA levels were found to be markedly reduced by 5-fold respectively in Ang II infused rats heart as compared with sham rat hearts. Moreover, cGMP production in response to ANP was reduced by 77% in the isolated cardiac membrane preparation from the Ang II infused rat hearts. Losartan treatment reversed NPR-A expression and responsiveness to ANP. This study suggests that Ang II down regulates cardiac NPR-A activity by suppressing Npr1 gene transcription.

Tyrosine phosphorylation of the human guanylyl cyclase C receptor.

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

Cellular signaling with nitric oxide and cyclic GMP

During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

Differentially-altered vascular guanylate cyclase isoforms in experimental hypertensive rats

Pathophysiological implications of the vascular nitric oxide (NO)/cGMP pathway were investigated in various rat models of hypertension. The expression of brain and endothelial constitutive NO synthases (bNOS, ecNOS) was determined by Western blot analysis, and the biochemical activity of soluble and particulate guanylate cyclases (GC) was assessed by the amount of cGMP generated in the thoracic aortae of rats with deoxycorticosterone acetate (DOCA)-salt, two-kidney, one dip (2K1C), and spontaneous hypertension (SHR). Plasma nitrite/ nitrate levels were decreased in DOCA-salt and 2K1C hypertension, and increased in SHR. The vascular expression of bNOS as well as that of ecNOS was decreased along with tissue nitrite/nitrate contents in DOCA-salt and 2K1C hypertension. The expression of both bNOS and ecNOS was increased in SHR with concomitant changes of tissue nitrite/nitrate contents. The activity of soluble GC was decreased, and that of particulate GC was increased in DOCA-salt hypertension. The soluble GC activity was increased, while the particulate GC activity was not affected in 2K1C hypertension. The soluble GC activity was not significantly changed, but the particulate GC activity was decreased in SHR. These results indicate that the high blood pressure is associated with differentially-altered vascular NO/cGMP pathway in different models of hypertension.

The effects of oxygen radicals on the activity of nitric oxide synthase and guanylate cyclase

Reactive oxygen species such as superoxides, hydrogen peroxide (H2O2) and hydroxyl radicals have been suggested to be involved in the catalytic action of nitric oxide synthase (NOS) to produce NO from L-arginine. An examination was conducted on the effects of oxygen radical scavengers and oxygen radical-generating systems on the activity of neuronal NOS and guanylate cyclase (GC) in rat brains and NOS from the activated murine macrophage cell line J774. Catalase and superoxide dismutase (SOD) showed no significant effects on NOS or GC activity. Nitroblue tetrazolium (NBT, known as a superoxide radical scavenger) and peroxidase (POD) inhibited NOS, but their inhibitory actions were removed by increasing the concentration of arginine or NADPH respectively, in the reaction mixture. NOS and NO-dependent GC were inactivated by ascorbate/FeSO4 (a metal-catalyzed oxidation system), 2'2'-azobis-amidinopropane (a peroxy radical producer), and xanthine/xanthine oxidase (a superoxide generating system). The effects of oxygen radicals or antioxidants on the two isoforms of NOS were almost similar. However, H2O2 activated GC in a dose-dependent manner from 100 microM to 1 mM without significant effects on NOS. H2O2-induced GC activation was blocked by catalase. These results suggested that oxygen radicals inhibited NOS and GC, but H2O2 could activate GC directly.

Guanylate cyclase activity in retina optic nerve and optic chiasm of rats under different illumination conditions

In the present work we have measured the guanylase cyclase activity in soluble fractions from several tissues relevant to the visual response under different illumination conditions. Guanylate cyclase was sensitive to changes of light / dark periods in incubated extract obtained from soluble fractions of retina, optic nerve and optic chiasm. The changes in soluble guanylate cylcase activity found, about 100 fold between dark and light periods in those tissues, indicate a key role for this enzyme. The results showed that light inhibit strongly the soluble retinal guanylate cyclase activity; while it increases the activity of this enzyme in the optic nerve. A generalized photoinhibited response of soluble guanylate cyclase eas observed in all studied tissues in prolonged dark adapted animals. The effect of Na+ 1 and 10 nM, and free Ca++ 28 M and 2.8 MuM on the guanylate cyclase activity was performed in the studied tissues. The enzymatic activity appeared to be inversely related in the retina and optic nerve with regard to the ion exposue, which may involve different ionic control mechanisms. All indicate an active role for the soluble guanylate cyclase in the phototransduction process not only in retina, also in other tissues relevant in the visual response.