Neuronal Nitric Oxide Reduces Sympathetic Excitability by Modulation of Central Glutamate Effects in Pigs

General Procedures

Experiments were performed on pigs (16 to 20 kg body weight) that were initially sedated by ketamine (20 mg/kg IM). Anesthesia was maintained by isoflurane (0.6% to 1.2%) in the inspired air, consisting of 2:1 N₂O/O₂. For infusion of drugs and for measurement of BP, catheters were placed into a femoral vein and artery (advanced in the abdominal aorta) and in the right atrium, respectively. Antibiotics (100 mg/kg ampicillin) were given to prevent possible influences of infections, such as the induction of expression of inducible NOS by bacterial endotoxins. The pigs were paralyzed by 0.2 mg/kg per hour pancuronium bromide and artificially ventilated via a tracheal tube. End-tidal CO₂ was kept at normal levels by adjustment of ventilatory depth and rate. Arterial blood gases were monitored with a blood gas analyzer (AVL 990, AVL List) and maintained in the normal range by administration of sodium bicarbonate solutions or adjustment of ventilation. Rectal temperature was maintained at 38.5°C by a thermostatically controlled infrared lamp. All pigs were bilaterally vagotomized. In addition, five pigs were completely baroreceptor-denervated by cutting both carotid sinus nerves after the vagotomy. For measurements of CO, a biluminal 5F Swan-Ganz thermodilution catheter (Baxter) was inserted through a jugular vein and advanced through the right ventricle in the arteria pulmonalis under BP control in 10 animals. In 8 other pigs, active redirection transient time flow probes (ART, Triton) were placed around the pulmonary artery for continuous measurement of CO. For this purpose, a thoracotomy was performed in the left fourth intercostal space. The chest was closed before the measurements. The similarity of CO data was checked by simultaneous measurements using both methods in separate experiments (data not shown). For recording of RSNA, the left renal nerve was retroperitoneally exposed, placed on bipolar platinum electrodes, and kept in a mixture of Vaseline and paraffin oil. Neural signals were amplified (×20 000 to ×50 000, Tektronix AM 502), filtered (2 Hz to 3 kHz), and stored and analyzed with a CED 1401 interface connected to an 80486 PC computer. RSNA was full wave–rectified, and then RC-integrated with a time constant between 7 and 10 milliseconds. To activate somatosympathetic reflex responses, the left great sciatic nerve (nervus ischiadicus) was placed on bipolar platinum electrodes, embedded in Vaseline, and connected to an isolated stimulator (Digitimer). Stimulation was performed to produce reproducible submaximal excitatory effects on RSNA using consecutive trains of 20-second length using 10 to 20 V, 30 Hz, and 0.5- to 1-millisecond pulse duration at intervals of 120 seconds.

Drugs and Infusions

7-NI and MeTC were from Alexis Chemicals. All other drugs were from Sigma Chemical Co. 7-NI was dissolved in DMSO. All other drugs were dissolved in distilled water. For preparation of the final concentrations, the substances were further diluted in Ringer’s solution. For ICV administrations, a catheter was inserted into the cerebroventricular space from the dorsal surface of the medulla at the level of the obex and advanced to the ventral surface of the medulla. The position of the catheter was functionally verified by instantaneous excitatory sympathetic responses to injections (0.3 mL) of glutamate, which are finally integrated within the brain stem by pre-SPNs within the RVLM.³ Central NOS inhibition was carried out by short-term infusion (within 5 minutes, 1 mL/min) of 7-NI (1 mmol/L, 5% DMSO), MeTC (0.1 mmol/L), or L-NNA (0.3 mmol/L). The reversibility of the effects of NOS inhibition was tested by short-term infusion of l-arginine (3.0 mmol/L) or SNAP (100 μmol/L) after NOS inhibition. As a test for central sympathetic excitability, 0.5 mL of 0.1 mol/L glutamate was injected (ICV) subsequent to the above pretreatments or sham control. Systemically, inhibition of NOS was carried out by 7-NI and/or L-NAME at doses of 20 mg/kg IV, respectively. The NO donor SNAP was infused systemically at a rate of 2 μg/kg per minute.

The effects of the solvent of 7-NI (ie, DMSO, 5%) were tested in 4 pigs. Infusion (ICV) of DMSO had no direct effects on RSNA and MAP but transiently increased CO (change, 0.37±0.16 L/min) and HR (change, 35±9 bpm). Furthermore, the responses to sciatic nerve stimulation of RSNA (298±21% versus 235±16%) and MAP (change, 27.4±5.2 versus 18.2±6.3 mm Hg) but not those to glutamate (ICV) were significantly increased by DMSO. The duration of these DMSO effects was 3 to 6 minutes. Thereafter, baseline RSNA and hemodynamics and responses to nerve stimulation returned to normal values. To prevent influences of DMSO on other experiments, we observed a period of at least 10 minutes before measurements after 7-NI treatments.

Data Analysis

HR was derived from the BP signal. TPR was calculated as follows: (MAP−CVP)/CO, expressed in mm Hg·L⁻¹·min⁻¹. RSNA was RC-integrated and measured in aU. For presentation in summarizing figures and for statistical evaluation, these data were normalized and expressed in percent of control values (% RSNA). All direct measurements were stored on a linear recorder (Gould) and on DAT tape (Sony DT-120RN) for further computing. Responses to drugs were separately calculated as individual differences from pretreatment values. Responses to electrical stimulation were calculated as average peak values derived from three consecutive stimuli, respectively. All data were analyzed by ANOVA (for repeated measurements where appropriate). Comparison of means was carried out with Tukey’s studentized range test. Differences of P<.05 were considered to be significant. Values are reported as mean±SEM.

Effects of ICV and Systemic Inhibition of NOS on Baseline Values

Changes in baseline RSNA and hemodynamics in response to ICV and systemic inhibition of nNOS by 7-NI and of all NOS isoforms by L-NAME are listed in Table 1⇓. 7-NI had no significant effects on RSNA either after ICV or systemic administration but caused significant increases in HR upon ICV administration. MeTC, which was administered (ICV) in five other pigs increased RSNA (to 132±12% of control, P<.05), slightly increased CO (from 2.65±0.18 to 3.01±0.22 L/min), but had no significant effect on MAP (from 103.6±7.4 to 92.0±8.1 mm Hg) or HR (from 131±11 to 143±13 bpm). In contrast, systemic blockade of all NOS isoforms resulted in large increases in TPR and BP and reductions in CO that were associated with decreased RSNA (Table 1⇓). The ICV administration of SNAP counteracted all of the effects of nNOS blockade except for the increases in HR caused by ICV administration of 7-NI.

Modulation of Glutamate Responses

Original recordings of the responses to glutamate (ICV) before and after ICV inhibition of all NOS isoforms by L-NNA (0.3 mmol/L) and subsequent to the administration of the NO donor SNAP (100 μmol/L ICV) are shown in Fig 1⇓. Glutamate caused reproducible sympathoexcitatory responses with latencies to onset of excitation between 10 and 30 seconds. The duration of the responses was 4 to 8 minutes. Inhibition of NOS increased the responses to glutamate, whereas SNAP attenuated them below control values. Fig 2A⇓ shows mean data for the changes in RSNA and MAP caused by glutamate before and after inhibition of nNOS by 7-NI compared with the effects of global NOS inhibition and the effect of subsequent treatment by the S-nitrosothiol SNAP. The correspondent hemodynamic changes are shown in Table 2⇓.

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Figure 1.

Variation of glutamate responses by NO. Original tracings show changes in BP, RSNA, and HR. L-NNA (0.3 mmol/L) and SNAP (100 μmol/L) were administered by short-term ICV infusion. Control indicates sham infusion (Ringer’s solution). Glutamate responses were evoked by intracerebroventricular (i.c.v.) injection of 0.5 mL of 0.1 mol/L glutamate.

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Figure 2.

Modulation of the pressor effects (ΔMAP) and sympathoexcitatory responses (% RSNA) to glutamate by NO within the brain stem. Glutamate tests and pretreatments were carried out as described in Fig 1⇑. A, Responses to glutamate following pretreatments with sham control, 7-NI (1 mmol/L), L-NNA (0.3 mmol/L), and SNAP (100 μmol/L) (n=7). B, Responses to glutamate following pretreatments with sham control, MeTC (0.1 mmol/L), and l-arginine (L-Arg, 3.0 mmol/L) (n=5). *P<.05, **P<.01 vs control values.

The responses of RSNA and MAP to glutamate before and after ICV inhibition of nNOS by MeTC and subsequent to the additional treatments by l-arginine and SNAP are shown in Fig 2B⇑. The correspondent hemodynamic responses are listed in Table 3⇓. MeTC and 7-NI had qualitatively similar effects on the responses to glutamate. After the recovery of endogenous nNOS activity by l-arginine, almost normal responses to glutamate were observed. Nevertheless, SNAP also caused further inhibition of glutamate responses in the experiments with MeTC.

Modulation of Somatosympathetic Reflexes

Examples of the effects of ICV administration of MeTC and l-arginine after MeTC on the responses to sciatic nerve stimulation are shown in Fig 3⇓. The mean data for stimulation experiments with 7-NI and MeTC are summarized in Fig 4⇓. The respective hemodynamic data are summarized in Tables 2⇑ and 3⇑. Inhibition of neuronal NOS by both compounds potentiated somatosympathetic reflex responses significantly. Systemic inhibition of nNOS by 7-NI or of all NOS isoforms by L-NNA/L-NAME had no significant additional effects. Similarly, when L-NNA/L-NAME was given first, there were no further neuronal or hemodynamic effects of ICV or intravenous 7-NI (n=3 each, data not shown). Effects of nNOS inhibition by MeTC were significantly reduced by l-arginine and antagonized even more pronouncedly by the S-nitrosothiol SNAP, which was similarly effective in the experiments with 7-NI or L-NNA/L-NAME.

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Figure 3.

Effects of NO on somatosympathetic reflex responses. Original tracings show changes in BP, RSNA, and HR in response to electrical stimulation of the left greater sciatic nerve (nervus ischiadicus) in a representative pig. Pretreatments by sham control and intracerebroventricular (i.c.v.) MeTC, and l-arginine are as described in Figs 1⇑ and 2⇑.

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Figure 4.

Effects of intracerebroventricular (i.c.v.) or intravenous (i.v.) treatment with sham control, 7-NI (1 mmol/L i.c.v., 20 mg/kg i.v.), L-NNA (0.3 mmol/L i.c.v.), L-NAME (20 mg/kg i.v.), and SNAP (100 μmol/L i.c.v., 2 μg·kg⁻¹·min⁻¹ i.v.) (n=8, i.c.v.; n=6, i.v.) (A) or sham control, MeTC (0.1 mmol/L i.c.v.), l-arginine (L-Arg, 3.0 mmol/L i.c.v.), and SNAP (100 μmol/L i.c.v.) (n=5) (B) on the responses of mean BP (ΔMAP) and RSNA (% RSNA) to electrical stimulation of the left greater sciatic nerve (10 to 20 V, 1-millisecond pulse duration, 30 Hz for 20 seconds every 2 minutes). *P<.05, **P<.01 vs control values.

Effects of Baroreceptor Denervation

To test whether inhibitory baroreflex inputs to the RVLM might influence the modulation of RSNA by NO, we performed similar experiments in completely baroreceptor-denervated animals (n=5). The results are summarized in Table 2⇑. Baseline RSNA (10.5±1.9 aU), CO (2.40±0.34 L/min), TPR (45.3±6.0 mm Hg·L⁻¹·min⁻¹), MAP (105.0±10.4 mm Hg), and HR (145±12 bpm) were not significantly different from those shown in Table 1⇑ for control pigs. The responses to glutamate are 126±14%, and those to electrical reflex stimulation represent 122±16% of the responses in control animals. These changes in RSNA were paralleled by analogue effects on MAP, CO, TPR, and HR (Table 2⇑). However, except for the increases in TPR, these changes were not statistically significant. Furthermore, there were no significant differences in the effects of NOS inhibition or SNAP on the responses to glutamate or sciatic nerve stimulation in these baroreceptor-denervated animals.