8-bromocyclic-gmp and 7-nitroindazole

The aim of this work was to investigate in the avascular heart of the frog Rana esculenta the influence of nitric oxide (NO) on ventricular systolic and diastolic functions by using a novel image analysis technique. The external volume variations of the whole ventricle were monitored during the heart cycle by video acquisition(visible light) and analysed by an appropriately developed software with a specific formula for irregular convex solids. The system, which measures the rate of volume changes and the ejection fraction, directly determined the volumetric behaviour of the working frog heart after stimulation or inhibition of NOS-NOcGMP pathway. End-diastolic volume (EDVext), end-systolic volume (ESVext), contraction and relaxation velocities (dV/dtsys and dV/dtdia, respectively), stroke volume (SV) and ejection fraction (EF), were measured before and after perfusion with NOS substrate (L-arginine), NO donor (SIN-1), cGMP analogue (8-Br-cGMP),NOS inhibitors (NG-monomethyl-L-arginine, L-NMMA; L-N(5)-(1-iminoethyl)-ornithine, L-NIO; 7-Nitroindazole,7-NI) and guanylyl cyclase inhibitor (ODQ). The results showed that NO reduces ventricular systolicfunction improving diastolic filling, while NOS inhibition increases contractility impairing ventricular filling capacity. The presence of activated eNOS (p-eNOS) was morphologically documented, further supporting that the mechanical activity of the ventricular pump in frog is influenced by a tonic release of NOS-generated NO.

Topics: Animals; Anura; Arginine; Cyclic GMP; Female; Heart Ventricles; In Vitro Techniques; Indazoles; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Perfusion; Serine; Software; Ventricular Function

It is known that several second messengers, such as Ca(2+) or cAMP, play important roles in the intracellular pathway of electrolyte secretion in tracheal submucosal gland. However, the participation of cGMP, and therefore nitric oxide (NO), is not well understood. To investigate the physiologic role of NO, we first examined whether tracheal glands can synthesize NO in response to acetylcholine (ACh), and then whether endogenous NO has some effects on the ACh-triggered ionic currents. From the experiments using the NO-specific fluorescent indicator 4,5-diaminofluorescein diacetate salt (DAF-2DA), we found that a physiologically relevant low dose of ACh (100 nM) stimulated the endogenous NO synthesis, and it was almost completely suppressed in the presence of the nonspecific NO synthase (NOS) inhibitor Nomega-Nitro-L-arginine Methyl Ester Hydrochloride (L-NAME) or the neuronal NOS (nNOS)-specific inhibitor 7-Nitroindazole (7-NI). Patch-clamp experiments revealed that both the NOS inhibitors (L-NAME or 7-NI) and cGK inhibitors (KT-5823 or Rp-8-Br-cGMP) partially decreased ionic currents induced by 30 nM of ACh, but not in the case of 300 nM of ACh. Our results indicate that NO can be synthesized through the activation of nNOS endogenously and has potentiating effects on the gland secretion, under a physiologically relevant ACh stimulation. When cells were stimulated by an inadequately potent dose of ACh, which caused an excess elevation in [Ca(2+)](i), the cells were desensitized. Therefore, due to NO, gland cells become more sensitive to calcium signaling and are able to maintain electrolyte secretion without desensitization.

Topics: Acetylcholine; Animals; Calcium Signaling; Carbazoles; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Exocrine Glands; In Vitro Techniques; Indazoles; Indoles; Models, Biological; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Protein Kinase Inhibitors; Sus scrofa; Trachea

The ability of endothelins 1 and 3 (ET-1 and ET-3) to reduce neuronal norepinephrine release through ETB receptor activation involving nitric oxide (NO) pathways in the rat anterior hypothalamus region (AHR) was previously reported. In the present work, we studied the effects of ET-1 and -3 on tyrosine hydroxylase (TH) activity and the possible involvement of NO pathways. Results showed that ET-1 and -3 (10 nM) diminished TH activity in AHR and this effect was blocked by a selective ETB receptor antagonist (100 nM BQ-788), but not by a ET(A) receptor antagonist (BQ-610). To confirm these results, 1 microM IRL-1620 (ET(B) agonist) reduced TH activity whereas 300 nM sarafotoxin S6b falled to modify it. N(omega)-Nitro-L-arginine methyl ester (10 microM), 7-nitroindazole (10 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-ona (10 microM), KT5823 (2 microM), inhibitors of nitric oxide synthase, neuronal nitric oxide synthase, NO-sensitive-guanylyl cyclase, and protein kinase G, respectively, did not modify the reduction of TH activity produced by ETs. In addition, both 100 microM sodium nitroprusside and 50 microM 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and guanosine-3',5'-cyclic monophosphate analog, respectively) diminished TH activity. Present results showed that ET-1 and ET-3 diminished TH activity through the activation of ET(B) receptors involving the NO/guanosine-3',5'-cyclic monophosphate/protein kinase G pathway. Taken jointly present and previous results it can be concluded that both ETs play an important role as modulators of norepinephrine neurotransmission in the rat AHR.

Topics: Animals; Carbazoles; Cyclic GMP; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-3; Hypothalamus, Anterior; Indazoles; Indoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Nitroprusside; Oligopeptides; Oxadiazoles; Piperidines; Quinoxalines; Rats; Suramin; Tyrosine 3-Monooxygenase

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.

Topics: Animals; Arachidonic Acid; Calcium; Calcium Signaling; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Fura-2; Gadolinium; Indazoles; Mice; Nitric Oxide; Parotid Gland; Ryanodine; Tetracaine; Thapsigargin; Triazoles

Endogenous opioids and nitric oxide (NO) are recognized modulators of cardiac function. Enkephalins and inhibitors of NO synthase (NOS) both produce similar interruptions in the vagal control of heart rate. This study was conducted to test the hypothesis that NO systems within the canine sinoatrial (SA) node facilitate local vagal transmission and that the endogenous enkephalin methionine-enkephalin-arginine-phenylalanine (MEAP) attenuates vagal bradycardia by interrupting the NOS-cGMP pathway. Microdialysis probes were inserted into the SA node, and they were perfused with nonselective (Nomega-nitro-l-arginine methyl ester) and neuronal (7-nitroindazole) NOS inhibitors. The right vagus nerve was stimulated and both inhibitors gradually attenuated the resulting vagal bradycardia. The specificity of these inhibitions was verified by an equally gradual reversal of the inhibition with an excess of the NOS substrate l-arginine. Introduction of MEAP into the nodal interstitium produced a quickly developing but quantitatively similar interruption of vagal bradycardia that was also slowly reversed by the addition of l-arginine and not by d-arginine. Additional support for convergence of opioid and NO pathways was provided when the vagolytic effects of MEAP were also reversed by the addition of the NO donor S-nitroso-N-acetyl-penicillamine, the protein kinase G activator 8-bromo-cGMP, or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. MEAP and 7-nitroindazole were individually combined with the direct acting muscarinic agonist methacholine to evaluate potential interactions with muscarinic receptors within the SA node. MEAP and 7-nitroindazole were unable to overcome the bradycardia produced by methacholine. These data suggest that NO and enkephalins moderate the vagal control of heart rate via interaction with converging systems that involve the regulation of cAMP within nodal parasympathetic nerve terminals.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Arginine; Bradycardia; Cyclic GMP; Dogs; Dose-Response Relationship, Drug; Enkephalin, Methionine; Enzyme Inhibitors; Indazoles; Methacholine Chloride; NG-Nitroarginine Methyl Ester; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Parasympathomimetics; Penicillamine; Phosphodiesterase Inhibitors; Sinoatrial Node; Vagus Nerve

1. A possible role of nitric oxide (NO) as a modulator of capsaicin-sensitive sensory neurotransmission in blood vessels was investigated in the rat isolated mesenteric arterial bed. 2. Electrical field stimulation (EFS) of methoxamine-preconstricted mesenteric beds elicited frequency-dependent vasorelaxation mediated by capsaicin-sensitive sensory nerves. N(G)-nitro-L-arginine methyl ester (L-NAME, 10 and 300 microM) and 7-nitroindazole (7-NI, 100 microM), inhibitors of nitric oxide synthase (NOS), augmented sensory neurogenic vasorelaxation. D-NAME (300 microM), 6-aminoindazole (100 microM) and N(omega)-propyl-L-arginine (50 nM), a selective inhibitor of neuronal NOS, were without effect. The effect of 10 microM L-NAME was reversed by L-arginine (1 mM), the substrate for NOS. 3. L-NAME (300 microM) and 7-NI (100 microM) had no significant effect on vasorelaxations to calcitonin gene-related peptide (CGRP), the principal motor neurotransmitter of capsaicin-sensitive sensory nerves in rat mesenteric arteries, or to capsaicin, indicating a prejunctional action. The inhibitors of NOS had no effect on vasorelaxation to forskolin, but augmented vasorelaxation to sodium nitroprusside (SNP). 4. Removal of the endothelium augmented sensory neurogenic vasorelaxation, but did not affect vasorelaxation to CGRP, indicating a prejunctional action of endothelial NO. 5. In the absence of endothelium, L-NAME (300 microM) inhibited, and 7-NI (100 microM) caused no further augmentation of sensory neurotransmission. 6. SNP (100 nM), a nitric oxide donor, attenuated sensory neurogenic relaxations to EFS. 7. In rat isolated thoracic aortic rings, L-NAME (100 microM) and 7-NI (100 microM) attenuated concentration-dependent relaxations to acetylcholine. 8. These data show that NO modulates sensory neurotransmission evoked by EFS of the rat isolated mesenteric arterial bed, and that when NO synthesis is blocked sensory neurogenic relaxation is augmented. The source of NO is the vascular endothelium.

Topics: Animals; Arginine; Bucladesine; Calcitonin Gene-Related Peptide; Capsaicin; Cyclic GMP; Electric Stimulation; Endothelium, Vascular; In Vitro Techniques; Indazoles; Male; Mesenteric Arteries; Neurons, Afferent; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitroprusside; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.

Topics: Animals; Benzoates; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Indazoles; Kidney; Male; Nervous System; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroarginine; Norepinephrine; Oxadiazoles; Oxazines; Perfusion; Pressure; Rats; Rats, Wistar

The grasshopper embryo has been used as a convenient system with which to investigate mechanisms of axonal navigation and pathway formation at the level of individual nerve cells. Here, we focus on the developing antenna of the grasshopper embryo (Schistocerca gregaria) where two siblings of pioneer neurons establish the first two axonal pathways to the CNS. Using immunocytochemistry we detected nitric oxide (NO)-induced synthesis of cGMP in the pioneer neurons of the embryonic antenna. A potential source of NO are NADPH-diaphorase-stained epithelial cells close to the basal lamina. To investigate the role of the NO/cGMP signaling system during pathfinding, we examined the pattern of outgrowing pioneer neurons in embryo culture. Pharmacological inhibition of soluble guanylyl cyclase (sGC) and of NO synthase (NOS) resulted in an abnormal pattern of pathway formation in the antenna. Axonogenesis of both pairs of pioneers was inhibited when specific NOS or sGC inhibitors were added to the culture medium; the observed effects include the loss axon emergence as well as retardation of outgrowth, such that growth cones do not reach the CNS. The addition of membrane-permeant cGMP or a direct activator of the sGC enzyme to the culture medium completely rescued the phenotype resulting from the block of NO/cGMP signaling. These results indicate that NO/cGMP signaling is involved in axonal elongation of pioneer neurons in the antenna of the grasshopper.

Topics: Animals; Axonal Transport; Axons; Cyclic GMP; Dihydrolipoamide Dehydrogenase; Embryo, Nonmammalian; Enzyme Inhibitors; Grasshoppers; Guanylate Cyclase; Horseradish Peroxidase; Indazoles; Morphogenesis; Nervous System; Neurons; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Quinoxalines; Signal Transduction

Previous experimental findings have led to the suggestion that guanosine 3',5'-cyclic monophosphate (cGMP) plays a permissive role in hypercapnic cerebral vasodilation. However, we recently reported that the technique used to reveal a permissive role for cGMP [cGMP repletion in the presence of nitric oxide synthase (NOS) inhibition] created a situation where CO(2) reactivity was normalized but where different mechanisms (i.e., K(+) channels) participated in the response. In the present study, we examined whether that nascent K(+)-channel dependence is related in any way to an increase in the influence of the miconazole-inhibitable cytochrome P-450 epoxygenase pathway. Using intravital microscopy and a closed cranial window system in adult rats, we measured pial arteriolar diameters during normo- and hypercapnia, first in the absence and then in the presence of a neuronal NOS (nNOS) inhibitor [7-nitroindazole (7-NI)]. This was followed by suffusion of a cGMP analog and then cGMP plus miconazole. Separate groups of rats were used to evaluate whether miconazole either alone or in the presence of 8-bromoguanosine 3', 5'-cyclic monophosphate (8-BrcGMP) or its vehicle (0.1% ethanol) had any effect on CO(2) reactivity and whether miconazole affected K(+)-channel opener-induced dilations. Hypercapnic (arterial PCO(2), congruent with65 mmHg) pial arteriolar dilations, as expected, were reduced by 70-80% with 7-NI and restored with cGMP repletion. CO(2) reactivity was again attenuated after miconazole introduction. Miconazole, with and without 8-BrcGMP, and its vehicle had no influence on pial arteriolar CO(2) reactivity in the absence of nNOS inhibition combined with cGMP repletion. Miconazole alone also did not affect vasodilatory responses to K(+)-channel openers. Thus present results suggest that the nascent K(+)-channel dependence of the hypercapnic response found in our earlier study may be related to increased epoxygenase activity. The specific reasons why the pial arteriolar CO(2) reactivity gains a K(+)-channel and epoxygenase dependence only under conditions of nNOS inhibition and cGMP restoration remain to be identified. These findings again call into question the interpretations applied to data collected in studies evaluating potential permissive actions of cGMP or NO.

Topics: Animals; Arterioles; Carbon Dioxide; Cyclic GMP; Drug Combinations; Enzyme Inhibitors; Hypercapnia; Indazoles; Miconazole; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Pia Mater; Potassium Channels; Rats; Rats, Sprague-Dawley; Reference Values; Vasodilation

The conclusion that cyclic 3'-5 guanosine monophosphate (cGMP) functions in a 'permissive' manner in promoting cerebrovasodilation during hypercapnia was based on findings showing that the nitric oxide synthase (NOS) inhibitor-induced repression of the CO2 response could be reversed upon addition of exogenous cGMP. We hypothesized that the action of cGMP revealed in those studies does not define its normal role in hypercapnic cerebral vasodilation, but rather is a unique function of the artificial situation of NOS inhibition coupled with cGMP repletion. Thus, although CO2 reactivity may be the same in normal versus cGMP-repleted animals, the factors contributing to that response may differ. To test that possibility, the effects of calcium-dependent (KCa) or ATP-sensitive (KATP) potassium channel blockers on pial arteriolar CO2 reactivity, in vivo, were evaluated in the presence and absence of NOS inhibition plus administration of a cGMP analogue. Pial arteriolar diameter changes in hypercapnia were measured in three principal groups of anesthetized rats: (I) KCa channel-inhibited (via iberiotoxin); (II) KATP channel-inhibited (via glibenclamide); and (III) controls. Group I and II rats were further divided into: (a) those treated with the neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI), followed by successive suffusions of the cGMP analogue, 8-bromo-cGMP (8Br-cGMP) and 8Br-cGMP+K-channel blocker; and (b) rats where 7-NI and 8Br-cGMP applications were omitted. Group III rats were divided into time and 8Br-cGMP controls. Hypercapnia (PCO2 congruent with60 mmHg, 3 min)-induced dilations were reduced by 70-80% following 7-NI and restored by 8Br-cGMP. That restoration was reversed by both K-channel blockers. In the absence of 7-NI and exogenous cGMP, CO2 reactivity was unaffected by K-channel inhibition. These findings confirmed that nNOS-derived NO is critically important to the hypercapnic reactivity of cerebral arterioles, and that cGMP repletion, following NOS inhibition, could restore CO2 reactivity. The observation that KCa and KATP channel blockade did not alter CO2 reactivity under baseline conditions, but attenuated CO2 reactivity only in the presence nNOS inhibition (and cGMP repletion), suggests that multiple, redundant, and interactive mechanisms participate in CO2-induced vasodilation. These results also imply that current strategies for revealing permissive actions of cGMP (or NO) may need to be re-evaluated.

Topics: Adenosine Triphosphate; Animals; Benzimidazoles; Blood Pressure; Calcium; Carbon Dioxide; Cerebral Arteries; Cromakalim; Cyclic GMP; Glyburide; Hydrogen-Ion Concentration; Hypercapnia; Indazoles; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Penicillamine; Peptides; Pia Mater; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley; Substrate Specificity; Vasodilation

Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.

Topics: Animals; Arousal; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; GABA Modulators; Indazoles; Male; Nitric Oxide; Nitric Oxide Synthase; Pentobarbital; Phosphorylation; Rats; Rats, Wistar; Thalamus

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

Topics: Animals; Blood Pressure; Carbon Dioxide; Cerebellum; Cyclic GMP; Electric Stimulation; Enzyme Inhibitors; Glutamic Acid; Hyperemia; Indazoles; Male; Molsidomine; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxygen; Papaverine; Partial Pressure; Penicillamine; Rats; Rats, Sprague-Dawley; Regional Blood Flow; S-Nitroso-N-Acetylpenicillamine; Vasodilation