Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii

The anatomical relationship between vagal afferents and brain nitric oxide synthase containing terminals in the nucleus tractus solitarii was studied by means of anterograde tracing combined with immunocytochemistry and immuno-electron microscopy. Biotinylated dextran amine was injected into the nodose ganglion with a glass micropipette. Four to eight days following the injection, regions of the nucleus tractus solitarii containing biotinylated dextran amine-labelled vagal afferents and those containing nitric oxide synthase-immunopositive terminals were congruent. Many neurons exhibiting nitric oxide synthase immunoreactivity were found within the biotinylated dextran amine-containing terminal field. However dense labeling of terminals with biotinylated dextran amine precluded determination if the terminals were nitric oxide synthase-immunoreactive. Therefore, we combined degeneration of vagal afferents after removal of one nodose ganglion with nitric oxide synthase immuno-electron microscopy. Axon terminals that possessed characteristic vesicle clusters and were partially or completely engulfed by glial processes were identified as degenerating vagal afferents. Degenerating axon terminals comprised 38% of the total axon terminals in the nucleus tractus solitarii in a sample of sections; and of the degenerating axon terminals, 67% were nitric oxide synthase-immunoreactive. Nitric oxide synthase immunoreactivity was present in 41% of the non-degenerating axon terminals. Prominent staining of dendrites for nitric oxide synthase immunoreactivity indicated that much of the nitric oxide synthase in the nucleus tractus solitarii is not derived from peripheral afferents. Of the total number of dendritic profiles sampled, half were nitric oxide synthase-immunoreactive. Our data support the hypothesis that nitric oxide or nitric oxide donors may be present in primary vagal afferents that terminate in the nucleus tractus solitarii. While this study confirms that vagal afferents contain brain nitric oxide synthase, it demonstrates for the first time that the majority of nitric oxide synthase immunoreactivity in the nucleus tractus solitarii is found in intrinsic structures in the nucleus. In addition, our data show that second or higher order neurons in the nucleus tractus solitarii may be nitroxidergic and receive both nitroxidergic and non-nitroxidergic vagal input.     

Prostaglandin E2 facilitates excitatory synaptic transmission in the nucleus tractus solitarii of rats

Intrinsic membrane properties and synaptic responses of neocortical neurons located lateral to photochemically induced ischemic lesions were investigated using neocortical slice preparation. In comparison to neurons from control slices, these neurons had a significantly less negative resting membrane potential without any significant change in input resistance. In addition, gamma-aminobutyric acid (GABA) mediated synaptic inhibition was found to be less efficient; the conductances of both the early and late inhibitory postsynaptic potentials (IPSPs) were significantly smaller, and the reversal potential of the early IPSP was shifted to a more positive value. In some of the neurons, 'epileptiform' postsynaptic potentials could be elicited, which were abolished after wash-in of the N-methyl-D-aspartic acid (NMDA)-receptor antagonist D-2-amino-5-phosphonovaleric acid (AP-5). The results provide a possible explanation for the hyperexcitability found in the vicinity of cortical infarcts.     

Cerebral extracellular lactate concentration and blood flow during chemical stimulation of the nucleus tractus solitarii in anesthetized rats

The extracellular lactate concentration and blood flow in the cerebral cortex of urethane-anesthetized, paralyzed and artificially ventilated rats were monitored continuously and simultaneously using an enzyme electrode and a laser Doppler flowmeter (LDF), respectively, during chemical stimulation of the nucleus tractus solitarii (NTS) by microinjection of L-glutamate (1.7 nmol 50 nl). Chemical stimulation of the NTS significantly decreased the arterial blood pressure (ABP) from 85 +/- 17 to 68 +/- 14 mmHg, heart rate from 418 +/- 13 to 402 +/- 19 beats x min(-1) and cerebral blood flow (CBF) by 17.9 +/- 6.2% (P < 0.001). However, chemical stimulation of the NTS significantly increased the lactate concentration by 58.9 +/- 17.3 microM (P < 0.001). Barostat maneuver, which held systemic ABP constant during chemical stimulation of the NTS attenuated the responses in CBF and lactate concentration by 30 and 27%, respectively. The onset of the increase in lactate concentration was delayed about 19 s after that of the CBF decrease. Circulatory lactate produced no significant change in the cerebral extracellular lactate concentration. These results indicate that chemical stimulation of the NTS induces an increase in extracellular lactate concentration in the cerebral cortex through a decrease in CBF via cerebral vasoconstriction.     

Glutamate, gamma-aminobutyric acid and tachykinin-immunoreactive synapses in the cat nucleus tractus solitarii

Neurophysiological and pharmacological evidence suggests that glutamate, gamma-aminobutyric acid and tachykinins (substance P and neurokinin A) each have a role in cardiovascular regulation in the nucleus tractus solitarii. This study describes the ultrastructural relationships between nerve terminals immunoreactive for these substances in the nucleus tractus solitarii of the cat using post-embedding immunogold (single and double) labelling techniques on sections of tissue embedded in LR White resin. The technique combines a high specificity of labelling with good ultrastructural and antigenic preservation. Glutamate-immunoreactive terminals, recognized by their high density of gold particle labelling compared to the mean tissue level of labelling, accounted for about 40% of all synaptic terminals in the region of the nucleus tractus solitarii analysed (medial, dorsal, interstitial, gelatinosus and dorsolateral subnuclei). They appeared to comprise several morphological types, but formed mainly asymmetrical synapses, most often with dendrites of varying size, and contained spherical clear vesicles together with fewer dense-cored vesicles. Substance P- and neurokinin A-immunoreactive terminals were fewer in number (9% of all terminals) but similar in appearance, with the immunoreaction restricted to the dense-cored vesicles. Analysis of serial- and double-labelled sections showed a co-existence of substance P and neurokinin A-immunoreactivity in 21% of glutamate-immunoreactive terminals. Immunoreactivity for gamma-aminobutyric acid was found in 33% of all terminals in the nucleus tractus solitarii. These predominantly contained pleomorphic vesicles and formed symmetrical synapses on dendrites and somata. Possible sites of axo-axonic contact by gamma-aminobutyric acid-immunoreactive terminals onto glutamate-or tachykinin-immunoreactive terminals were rare, but examples of adjacent glutamate and gamma-aminobutyric acid-immunoreactive terminals synapsing on the same dendritic profile were frequent. These results provide an anatomical basis for a gamma-aminobutyric acid mediated inhibition of glutamatergic excitatory inputs to the nucleus tractus solitarii at a post-synaptic level.     

Elevated Fos expression in the nucleus tractus solitarii is associated with reduced baroreflex response in spontaneously hypertensive rats

We delineated the functional role of Fos protein at the nucleus tractus solitarii in the manifestation of reduced baroreceptor reflex control of heart rate during hypertension, using spontaneously hypertensive rats (SHR), stroke-prone SHR, Wistar-Kyoto rats, or Sprague-Dawley rats. Microinjection into the bilateral nucleus tractus solitarii of an antisense oligonucleotide that targets against the initiation codon of c-fos mRNA significantly potentiated the baroreceptor reflex in response to 30 minutes of sustained increase in blood pressure. Of particular note was the restoration of both the impaired sensitivity and capacity of baroreceptor reflex in SHR and stroke-prone SHR to levels comparable to those in normotensive rats. Likewise, the number of Fos-immunoreactive nuclei evoked by the sustained increase in blood pressure in the caudal nucleus tractus solitarii of SHR and stroke-prone SHR was reduced, after this antisense c-fos treatment, to the basal level exhibited by the normotensive animals. Control treatment with the corresponding sense oligonucleotide, an antisense oligonucleotide that targets against a different portion of the coding sequence of the c-fos mRNA or artificial cerebrospinal fluid, on the other hand, elicited no discernible effect on either the baroreceptor reflex response or the induced expression of Fos protein in the nucleus tractus solitarii by baroreceptor activation. We also found that the basal level of Fos expression in the caudal nucleus tractus solitarii was significantly elevated in the SHR and stroke-prone SHR. Together, these novel findings suggest that an elevated expression of basal Fos protein in the NTS during hypertension may be associated with the dysfunction in baroreceptor reflex control of heart rate.     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on gamma-aminobutyric acid (GABA)-induced currents (IGABA) was also examined. 2. DM inhibited 30 microM glycine-induced current (IGly), without affecting the current caused by 30 microM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 microM, and reversible. The half-maximum inhibitory concentration (IC50) of DM was 3.3 microM, about 85 times higher than that of strychnine. 3. DM 3 microM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 microM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 microM shifted the curve for glycine in a nearly parallel fashion. 4. The effect of 10 microM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 microM, inhibited IGly equally at -50 mV and +50 mV. The effect of 3 microM DM on IGly showed no use-dependence. Blockade by strychnine 0.1 microM showed no voltage- or use-dependence. 5. The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl- channel of the complex.     

Cardiovascular effects of chronic inhibition of nitric oxide synthesis and dietary salt in spontaneously hypertensive rats

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.     

Role of nitric oxide in the nucleus of the solitary tract of rats

We have determined the role of nitric oxide (NO) in the nucleus of the solitary tract (NTS) of normotensive Wistar rats. The unilateral microinjection of Nomega-nitro-l-arginine methyl ester (10 nmol) to block the synthesis of NO into the NTS significantly decreased the arterial pressure, heart rate (HR) and renal sympathetic nerve activity (RSNA) (-19+/-2 mmHg, -23+/-5 beats/min, -30+/-2%, respectively). The microinjection of carboxy-2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (Carboxy PTIO) (trapper of NO; 0.1 nmol) into the NTS also decreased arterial pressure and RSNA. Conversely, the microinjection of Et2N[N(O)NO]Na (NOC 18) (NO donor; 10 nmol) caused increases in arterial pressure, HR and RSNA (+14+/-2 mmHg, +11+/-2 beats/min, +38+/-7%, respectively), which was inhibited by the pre-microinjection of Carboxy PTIO (0.1 nmol). On the other hand, not only l-arginine (10 nmol) but also d-arginine (10 nmol), which is inactive to produce NO, significantly decreased the arterial pressure and RSNA. These results suggest that (1) NO acts at the NTS to increase the arterial pressure and RSNA, and (2) the microinjection of l-arginine as well as d-arginine led to decreases in arterial pressure and RSNA that were not mediated by the formation of NO in the NTS.     

Autoradiographic determination of local cerebral blood flow and local cerebral glucose utilization during chemical stimulation of the nucleus tractus solitarii of anesthetized rats

This study was conducted to determine whether the decrease in cerebral blood flow (CBF) observed during chemical stimulation of the nucleus tractus solitarius (NTS) can be explained by a decrease in cerebral metabolism. In anesthetized (urethane and chloralose), paralyzed and artificially ventilated rats, neurons in the NTS were chemically stimulated by microinjection of L-glutamate. Local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCGU) were quantified in 43 brain structures by quantitative autoradiographic techniques using [14C]iodoantipyrine and 2-[14C]deoxyglucose, respectively. During chemical stimulation of the NTS (n = 6), LCBF decreased significantly in 32 of the 43 structures investigated when compared to either a control group with artificial cerebrospinal fluid injection (n = 6), or a controlled hemorrhage group (n = 5). In the controlled hemorrhage group, blood pressure was decreased to a degree comparable to that induced by microinjection of L-glutamate into the NTS. Mean blood flow of all structures investigated was significantly (P < 0.01) lower in the stimulation group than that in the control group and in the hemorrhage group. No significant differences in LCGU were observed between the three investigated groups in all structures examined except for an increase in LCGU in the chemically stimulated NTS site. It is concluded that the decrease in LCBF measured in most brain structures during chemical stimulation of the NTS is not caused by a decrease in LCGU in these structures and may therefore be explained by neurogenic influences on brain vessels.     

Environmental events that modify the catecholamine fluorescence of the A2 cell bodies in nucleus tractus solitarii.

Found that the fluorescence intensity of the catecholamine cell bodies of the A2 group--as determined by microfluorimetry--increased significantly due to exposure of male rats of the Sprague-Dawley-Ivanovas strain to the following environmental events: placement from the colony cage into a cold (4°C) room for a period of 10 min, isolation from the colony for 6-7 days, and satiation of grouped Ss by allowing access to sweetened milk for 15 min. The following events failed to affect the intensity of the cells of A2: reduction of the group colony size from 8 to 3 rats per colony for 6-7 days and presentation of water instead of milk after the rats had experienced 6-7 days of milk satiation. Results indicate that aversive as well as rewarding environmental events activate the catecholamine cells of A2. (19 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.     

Whole-cell analysis of ionic currents underlying the firing pattern of neurons in the gustatory zone of the nucleus tractus solitarii

1. Previous work from this laboratory has shown that rostral nucleus tractus solitarii (rNTS) neurons can be separated into four different classes on the basis of responses to a current injection paradigm consisting of membrane hyperpolarization immediately followed by a depolarizing pulse. These classes have been termed Group I, II, III, and IV neurons. The regular repetitive firing discharge pattern of Group I cells is changed into an irregular spike train by membrane hyperpolarization. Hyperpolarization of Group II neurons delays the firing discharge induced by depolarization. Hyperpolarization had the least effect on the discharge pattern of Group III neurons. The discharge pattern of Group IV neurons consisted of a short burst of spikes. We used whole-cell recordings and pharmacological channel blockers in an in vitro brain stem slice preparation to determine the ionic basis for the repetitive firing properties of rNTS neurons. 2. Application of 4-aminopyridine (4-AP, 1 mM) decreased input resistance and increased action potential duration in all groups of neurons. However, the discharge pattern of Group I, III, and IV neurons was either unaltered or slightly modified by 4-AP. In contrast the delay in firing of Group II cells induced by hyperpolarization was strongly reduced and in some cases completely suppressed by application of 4-AP. This suggests that a 4-AP-sensitive conductance primarily underlies the firing pattern of Group II cells. 3. Voltage-clamp recordings revealed that the delay in Group II neurons is due to a transient outward potassium current that is partially inactivated around the resting membrane potential. Hyperpolarization removed this inactivation, causing a delay in the firing of the cell. The potassium current was blocked by 4-AP. A similar current was occasionally seen in neurons of the other groups. On the basis of its voltage and pharmacological dependence this current was presumed to be an A-current (IKA). 4. Blockade of calcium currents by a low-calcium (0.5 mM) saline containing 2 mM Co2+ depressed the excitability of rNTS cells. For Group II neurons the delay in firing activity was increased. In the other groups the repetitive firing pattern was suppressed. In addition the amplitude of the afterhyperpolarization occurring after a short train of action potentials was substantially reduced. This indicates that calcium currents (ICa) and calcium-activated potassium currents (IKCa) contribute to the repetitive firing properties of rNTS neurons. 5. In about half of Group I, III, and IV neurons an additional property was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat

1. The aim of this study was to characterize adenosine receptors located in the nucleus tractus solitarius (NTS) that mediate decreases in blood pressure in the anaesthetized rat. To determine the adenosine receptor subtype involved, a range of selective agonists and antagonists were studied and their relative potencies evaluated. 2. The rank order of agonist potency in inducing decreases in diastolic blood pressure was N6-cyclopentyladenosine (CPA) > N6-cyclohexyladenosine (CHA) > N-ethyl-carboxamidoadenosine (NECA) > or = 2-phenylaminoadenosine (CV1808) > 2-p-(carboxyethyl)phenethylamino-5' N-ethylcarboxamidoadenosine (CGS 21680) > N6-(2-(4-aminophenyl)ethyl)-adenosine (APNEA). 3. The hypotensive action of CPA following microinjection into the NTS was antagonized by i.v. infusions (50 micrograms kg-1 min-1) of adenosine receptor antagonists, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), 8-phenyltheophylline (8-PT), 8-(p-sulphophenyl)theophylline (8-SPT), and 1,3-dipropyl-8-N-(2-diethylamino)ethyl)-N methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo) benzenesulphonamidexanthine (PD 115199). The antagonist potency order was DPCPX > PD115199 > or = 8-PT. Intravenous infusion of 8-SPT had no effect on blood pressure responses to microinjection of CPA into the NTS. 4. The results suggest that adenosine A1 receptors in the NTS mediate hypotensive responses in the anaesthetized rat preparation.     

Mediation by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors in the expression of Fos protein at the nucleus tractus solitarii in response to baroreceptor activation in the rat

The extent of variation in reptile field metabolism, and its causal bases, are poorly understood. We studied the energetics of the insectivorous lizard Callisaurus draconoides at a site in the California Desert (Desert Center) and at a site at the southern tip of the Baja Peninsula (Cabo San Lucas; hereafter, Cabo). Reproducing Callisaurus were smaller at Cabo than at Desert Center. The allometry of metabolism with body mass can account for most differences in whole-animal metabolism. There was no significant effect of sex or source population on mass-adjusted metabolic rate in the laboratory (resting metabolism, measured by closed-system respirometry) or in the field (field metabolism, measured with doubly labeled water). The mass-adjusted resting metabolism and field metabolism of gravid females and the field metabolism of juvenile lizards were not significantly different from those of nonreproductive adults. Temperature had a significant effect on resting metabolism (Q10 = 2.7); fed lizards had resting metabolism that was 22% higher than that of fasted lizards; field metabolism was positively correlated with growth rate in juveniles; and field metabolism of adults increased from spring to late summer at Desert Center by 25%, probably because of longer activity period length and slightly higher activity period body temperature. We calculated from water influx and field metabolism that juveniles allocated 18% of their metabolizable energy intake to growth and that most energy deposited into eggs was transferred from energy stores rather than ingested in the weeks prior to laying.     

Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.     

Discharge pattern of neurons in the nucleus tractus solitarii (NTS): its cardiac rhythm is modulated by firing rate of the neurons

In the nucleus tractus solitarii (NTS) neurons discharge in relation to cardiac rhythm. This cardiac rhythm exhibits various patterns designated as CRDPs (cardiac rhythmic discharge patterns). The CRDPs are estimated by post-event-time histograms (PETH) triggered by the R-waves of the ECG. Modulations of CRDPs appear as changes in the number and height of peaks in the PETHs. The amount of basic activity, which is not related to the cardiac cycle, alters CRDP. PETHs constructed during various phases of respiration reveal modulations of CRDPs within the respiratory cycle. As our previous work indicated, the NTS neurons exhibit typical reticular rhythms. In this paper we also found that the basic activity of NTS neurons was often changed by other influences for which no comparable patterns could be observed in other simultaneously acquired signals. When we constructed PETHs according to the activity level of the NTS neurons, i.e., firing level per cardiac cycle, modulations of CRDPs which were even stronger than respiratory or reticular rhythmical modulations became clear. The modulations of CRDPs caused by different origins were found to be present in the same neuron interlaced in time. The possible role played by these modulations of CRDPs in the coordination of different functional systems in the organism is discussed.     

Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide

OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis. DESIGN: Prospective, randomized, controlled study on anesthetized animals. SETTING: A cardiopulmonary research laboratory. SUBJECTS: Forty-seven male adult Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment. MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%). CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.     

GABAergic effects on nucleus tractus solitarius neurons receiving gastric vagal inputs

Single units in the region of the medial nucleus tractus solitarius (NTS), responding to electrical stimulation of gastric vagal fibers, were recorded in an in vitro neonatal rat brainstem-gastric preparation. gamma-Aminobutyric acid (GABA) subreceptor agonists and antagonists were applied to the gastric and brainstem compartments of the bath chamber to evaluate the peripheral gastric and central brainstem GABAergic effects on NTS neuronal activity. The gastric effects of the GABAA receptor agonist muscimol and GABAB receptor agonist baclofen were evaluated on 55 tonic units that received the gastric vagal inputs. For approximately 58% (32 of 55) and 38% (21 of 55) of the units observed, muscimol (30 microM; IC50 = 2.0 microM) and baclofen (30 microM; IC50 = 1.5 microM) in the gastric compartment induced a concentration-dependent inhibition of 36.2 +/- 3.1% (mean +/- S.E.) and 31.0 +/- 2.9% of the control level of the NTS neuronal activity, respectively. The brainstem effects of muscimol and baclofen were tested on 51 units. For approximately 90% (46 of 51) and 78% (40 of 51) of the units tested, muscimol (30 microM; IC50 = 1.3 microM) and baclofen (30 microM; IC50 = 1.1 microM) in the brainstem compartment produced a concentration-dependent inhibition of 54.1 +/- 3.4% and 48.9 +/- 3. 5% of the control level, respectively. The remaining NTS units were not affected by these two GABA agonists. Bicuculline (10 microM) and saclofen (10 microM), the GABAA and GABAB subreceptor antagonists, competitively antagonized the gastric and brainstem effects by muscimol and baclofen, respectively. Our results demonstrated that both GABAA and GABAB receptors in the stomach and brainstem play an important role in activity modulation of the medial NTS neurons receiving gastric vagal inputs in neonatal rats.     

Hemodynamic responses to endothelin-1 and endothelin antagonists microinjected into the nucleus tractus solitarius in rats

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5-2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N(G)-nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5-20 mg/kg, i.v.), but not by an inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1-100 microg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 microg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 microg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.