Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)]; transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20 mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH. (c) 2011 Elsevier Ltd. All rights reserved.

Role of the spinal cord NO/cGMP pathway in the control of arterial pressure and heart rate

The modulatory effect of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on sympathetic preganglionic neurons still deserves further investigation. The present study was designed to examine the role of the spinal cord NO/cGMP pathway in controlling mean arterial pressure and heart rate. We observed that intrathecal administration of the NO synthase inhibitor N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) causes an increase in mean arterial pressure but does not affect heart rate. Intrathecal administration of the soluble guanylyl cyclase inhibitor 1H-[1,2,4] Oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) does not change mean arterial pressure and heart rate. The precursor for NO synthesis, L-arginine, reduces both mean arterial pressure and heart rate while administration of ODQ before L-arginine impaired decreases in mean arterial pressure and heart rate. Administration of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5) after L-NAME does not affect increases in mean arterial pressure promoted by NO synthase inhibition. Although the hypotensive and bradycardic responses induced by intrathecal administration of L-arginine depend on cGMP, our results indicate that NO acts to tonically inhibit SPNs, independent of either cGMP or NMDA receptors.

Evidence of early involvement of matrix metalloproteinase-2 in lead-induced hypertension

Lead exposure increases blood pressure (BP) by unknown mechanisms. Many recent studies have shown the involvement of matrix metalloproteinases (MMPs) in hypertension, particularly MMP-2. In this work, we have examined whether MMP-2 levels increase with lead-induced increase in BP. We have also investigated whether doxycycline (an MMP inhibitor) affects these alterations. To this end, rats were exposed to lead (90 ppm) and treated with doxycycline or vehicle for 8 weeks. Similar aortic and whole blood lead levels were found in lead-exposed rats treated with either doxycycline or vehicle. Lead-induced increases in BP and aortic MMP-2 levels (activity, protein, and mRNA) were blunted by doxycycline. Doxycycline also prevented lead-induced increases in the MMP-2/TIMP-2 mRNA ratio. No significant changes in vascular reactivity or morphometric parameters were found. In conclusion, lead exposure increases BP and vascular MMP-2, which is blunted by doxycycline. This observation suggests that MMP-2 may play a role in lead-induced increases in BP.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Involvement of the heme oxygenase-carbon monoxide-cGMP pathway in the nociception induced by acute painful stimulus in rats

Heme oxygenase-carbon monoxide-cGMP (HO-CO-cGMP) pathway has been reported to be involved in peripheral and spinal modulation of inflammatory pain. However, the involvement of this pathway in the modulation of acute painful stimulus in the absence of inflammation remains unknown. Thus, we evaluated the involvement of the HO-CO-cGMP pathway in nociception by means the of analgesia index (AI) in the tail flick test. Rats underwent surgery for implantation of unilateral guide cannula directed toward the lateral ventricle and after the recovery period (5-7 days) were subjected to the measures of baseline tail flick test Animals were divided into groups to assess the effect of intracerebroventricular administration (i.c.v.) of the following compounds: ZnDPBG (HO inhibitor) or vehicle (Na(2)CO(3)), heme-lysinate (substrate overload) or vehicle (L-lysine), or the selective inhibitor of soluble guanilate cyclase ODQ or vehicle (DMSO 1%) following the administration of heme-lysinate or vehicle. Heme overload increased AI, indicating an antinociceptive role of the pathway. This response was attenuated by i.c.v. pretreatment with the HO inhibitor ZnDPBG. In addition, this effect was dependent on cGMP activity, since the pretreatment with ODQ blocked the increase in the AI. Because CO produces most of its actions via cGMP, these data strongly imply that CO is the HO product involved in the antinociceptive response. This modulation seems to be phasic rather than tonic, since i.c.v. treatment with ZnDPBG or ODQ did not alter the AI. Therefore, we provide evidence consistent with the notion that HO-CO-cGMP pathway plays a key phasic antinociceptive role modulating noninflammatory acute pain. (C) 2011 Elsevier B.V. All rights reserved.

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

Altered expression of neuronal nitric oxide synthase in weaver mutant mice

The weaver mouse represents the only genetic animal model of gradual nigrostriatal dopaminergic neurodegeneration which is proposed as a pathophysiological phenotype of Parkinson`s disease. The aim of the present study was to analyze the nitric oxide and dopaminergic systems in selected brain regions of homozygous weaver mice at different postnatal ages corresponding to specific stages of the dopamine loss. Structural deficits were evaluated by quantification of tyrosine hydroxylase and neuronal nitric oxide synthase-immunostaining in the cortex, striatum, accumbens nuclei, subthalamic nuclei, ventral tegmental area, and substantia nigra compacta of 10-day, 1- and 2-month-old wildtype and weaver mutant mice. The results confirmed the progressive loss of dopamine during the postnatal development in the adult weaver mainly affecting the substantia nigra pars compacta, striatum, and subthalamic nucleus and slightly affecting the accumbens nuclei and ventral tegmental area. A general decrease in neuronal nitric oxide synthase-immunostaining with age was revealed in both the weaver and wild-type mice, with the decrease being most pronounced in the weaver. In contrast, there was an increase in the substantia nigra pars compacta nitric oxide synthase-immunostaining and a decrease mainly in the subthalamic and accumbens nuclei of the 2-month-old weaver mutant. The decrease in the expression of nNOS may bear functional significance related to the process of aging. DA neurons from the substantia nigra directly modulate the activity of subthalamic nucleus neurons, and their loss may contribute to the abnormal activity of subthalamic nucleus neurons. Although the functional significance of these changes is not clear, it may represent plastic compensating adjustments resulting from the loss of dopamine innervation, highlighting a possible role of nitric oxide in this process. (C) 2010 Elsevier B.V. All rights reserved.

Central NO-cGMP pathway in thermoregulation and survival rate during polymicrobial sepsis

Sepsis induces production of inflammatory mediators such as nitric oxide (NO) and causes physiological alterations, including changes in body temperature (T(b)). We evaluated the involvement of the central NO cGMP pathway in thermoregulation during sepsis induced by cecal ligation and puncture (CLP), and analyzed its effect on survival rate. Male Wistar rats with a T(b) probe inserted in their abdomen were intracerebroventricularly injected with 1 mu L N(G)-nitro-L-arginine methyl ester (L-NAME, 250 mu g), a nonselective NO synthase (NOS) inhibitor; or aminoguanidine (250 mu g), an inducible NOS inhibitor; or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.25 mu g), a guanylate cyclase inhibitor. Thirty minutes after injection, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 2 groups for determination of T(b) for 24 h and assessment of survival during 3 days. The drop in T(b) seen in the CLP group was attenuated by pretreatment with the NOS inhibitors (p < 0.05) and blocked with ODQ. CLP rats pretreated with either of the inhibitors showed higher survival rates than vehicle injected groups (p < 0.05), and were even higher in the ODQ pretreated group. Our results showed that the effect of NOS inhibition on the hypothermic response to CLP is consistent with the role of nitrergic pathways in thermoregulation.

Nitric oxide modulation of methylphenidate-induced disruption of prepulse inhibition in Swiss mice

Drugs that facilitate dopaminergic neurotransmission induce cognitive and attentional deficits which include inability to filter sensory input measured by prepulse inhibition (PPI) Methylphenidate, an amphetamine analog is used in the treatment of attention deficit hyperactivity disorder Given that nitric oxide (NO) modulates dopamine effect our aim is to analyze the nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) inhibitors effect on PPI disruption induced by methylphenidate The inhibitors effects were compared to those produced by haloperidol and clozapine Male Swiss mice received a first I p. Injection (one hour before testing), of either saline, or N(G) nitro L-arginine (10, 40 or 90 mg/kg) or 7-Nitroindazole (3, 10, 30 or 60 mg/kg). or oxadiazolo-quinoxalin (5 or 10 mg/kg). or haloperidol (1 mg/kg), or clozapine (5 mg/kg) Thirty min later mice received the second injection of either saline or methylphenidate (20 or 30 mg/kg) or amphetamine (5 or 10 mg/kg). One group of mice received intracerebroventricular 7-Nitroindazole (50 or 100 nM) followed by systemic administration of saline or methylphenidate (30 mg/kg) The results revealed a methylphenidate dose-dependent disruption of PPI comparable to amphetamine. The effect was prevented by either nitric oxide synthase or guanilate cyclase inhibitors or clozapine or haloperidol In conclusion, methylphenidate induced a dose-dependent PPI disruption in Swiss mice modulated by dopamine and NO/sGC. The results corroborate the hypothesis of dopamine and NO interacting to modulate sensorimotor gating through central nervous system. It may be useful to understand methylphenidate and other psychostimulants effects (C) 2009 Elsevier B.V All rights reserved

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway

There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinson`s disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the L-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, L-NOARG reduced 6-OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain. (c) 2008 Elsevier B.V. All rights reserved.

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

I Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO; NO gas solution) and nitroxyl ion (NO-; from Angeli's salt). 2 The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 muM), concentration-dependently inhibited responses to all agents. 10 muM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3 The NO scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-indazoline-1-oxyl-3-oxide; 100 muM) and hydroxocobalamin (100 muM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4 The NO- inhibitor, L-cysteine (3 mm), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5 The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO and NO-. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P

Inhibition of rat platelet aggregation by the diazeniumdiolate nitric oxide donor MAHMA NONOate

1 Inhibition of rat platelet aggregation by the nitric oxide (NO) donor MAHMA NONOate (Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-l-ium-1,2-diolate) was investigated. The aims were to compare its anti-aggregatory effect with vasorelaxation, to determine the effects of the soluble guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-ajquinoxalin-1-one), and to investigate the possible role of activation of sarco-encloplasmic reticulum calcium-ATPase (SERCA), independent of soluble guanylate cyclase, using thapsigargin. 2 MAHMA NONOate concentration-dependently inhibited sub-maximal aggregation responses to collagen (2 - 10 mug ml(-1)) and adenosine diphosphate (ADP; 2 mum) in platelet rich plasma. It was (i) more effective at inhibiting aggregation induced by collagen than by ADP, and (ii) less potent at inhibiting platelet aggregation than relaxing rat pulmonary artery. 3 ODQ (10 mum) caused only a small shift (approximately half a log unit) in the concentration-response curve to MAHMA NONOate irrespective of the aggregating agent. 4 The NO-independent activator of soluble guanylate cyclase, YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzy] indazole; 1 - 100 mum), did not inhibit aggregation. The cGMP analogue, 8-pCPT-cGMP (8-(4-chlorophenylthio)guanosine 3'5' cyclic monophosphate; 0.1 - 1 mm), caused minimal inhibition. 5 On collagen-aggregated platelets responses to MAHMA NONOate (ODQ 10 PM present) were abolished by thapsigargin (200 nm). On ADP-aggregated platelets thapsigargin caused partial inhibition. 6 Results with S-nitrosoglutathione (GSNO) resembled those with MAHMA NONOate. Glyceryl trinitrate and sodium nitroprusside were poor inhibitors of aggregation. 7 Thus inhibition of rat platelet aggregation by MAHMA NONOate (like GSNO) is largely ODQ-resistant and, by implication, independent of soluble guanylate cyclase. A likely mechanism of inhibition is activation of SERCA.

(-)-CGP 12177 increases contractile force and hastens relaxation of human myocardial preparations through a propranolol-resistant state of the beta1-adrenoceptor

Two forms of the activated beta(1)-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant PI-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1 Hz. In right atrium from non-ailing hearts in the presence of 200 nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (-logEC(50)[M] 7.3+/-0.1, E-max 23+/-1% relative to maximal (-)-isoprenaline stimulation of beta(1)- and beta(2)-adrenoceptors, n=86 patients), shortening of the time to reach peak force (-logEC(50)[M] 7.4+/-0.1, E-max 37+/-5%, n=61 patients) and shortening of the time to reach 50% relaxation (t(50%), -logEC(50)[M] 7.3+/-0.1, E-max 33+/-2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-beta(1)-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (-logEC(50)[M] 7.7+/-0.1, E-max 68+/-6%, n=6 patients, P