Nitric oxide infused in the solitary tract nucleus blocks brain glucose retention induced by carotid chemoreceptor stimulation

Nitric Oxide 25 (2011) 387–395

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Nitric oxide infused in the solitary tract nucleus blocks brain glucose retention induced by carotid chemoreceptor stimulation Monica Lemus a, Sergio Montero a,b, Caridad Aurea Leal c, Eliseo Portilla-de Buen c, Sonia Luquin d, Joaquin Garcia-Estrada e, Valery Melnikov b, Elena de Alvarez-Buylla a,⇑ a

Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Ave. 25 de Julio s/n, Colima 28045, Mexico Facultad de Medicina, Universidad de Colima, Ave. 25 de Julio s/n, Colima 28045, Mexico División de Investigación Quirúrgica, Centro de Investigación Biomédica de Occidente, IMSS, Guadalajara, Mexico d Departamento de Neurociencias, Universidad de Guadalajara, Mexico e División de Neurociencias, Centro de Investigación Biomédica de Occidente, IMSS, Guadalajara, Mexico b c

a r t i c l e

i n f o

Article history: Received 12 May 2011 Revised 19 August 2011 Available online 28 September 2011 Keywords: Brain glucose retention Nitroxidergic drugs nNOS gene

a b s t r a c t Previous work has shown that the carotid body glomus cells can function as glucose sensors. The activation of these chemoreceptors, and of its afferent nucleus in the brainstem (solitary tract nucleus – STn), induces rapid changes in blood glucose levels and brain glucose retention. Nitric oxide (NO) in STn has been suggested to play a key role in the processing of baroreceptor signaling initiated in the carotid sinus [1]. However, the relationship between changes in NO in STn and carotid body induced glycemic changes has not been studied. Here we investigated in anesthetized rats how changes in brain glucose retention, induced by the local stimulation of carotid body chemoreceptors with sodium cyanide (NaCN), were affected by modulation of NO levels in STn. We found that NO donor sodium nitroprusside (SNP) micro-injected into STn completely blocked the brain glucose retention reflex induced by NaCN chemoreceptor stimulation. In contrast, NOS inhibitor Nx-nitro-L-arginine methyl ester (L-NAME) increased brain glucose retention reflex compared to controls or to SNP rats. Interestingly, carotid body stimulation doubled the expression of nNOS in STn, but had no effect in iNOS. NO in STn could function to terminate brain glucose retention induced by carotid body stimulation. The work indicates that NO and STn play key roles in the regulation of brain glucose retention. Ó 2011 Elsevier Inc. All rights reserved.

Introduction The brain obtains its metabolic energy almost exclusively from glucose. The levels of this carbohydrate reaching the brain are tightly regulated [2,3]. Glucose controls the secretion of hormones and activates neurons in the peripheral and central nervous system (CNS). In the CNS, glucose-sensitive neurons are present in the brainstem and the hypothalamus [4,5]. In the periphery, portohepatic neurons have been suggested to participate in the homeostatic responses to changes in glycemia [6]. The carotid body is situated at the bifurcation of the internal and external carotid arteries, strategically located to sense levels of pO2, pCO2, pH and osmolarity in blood entering the brain. Studies in our laboratory [7] and other [8] have shown that the carotid body glomus cells also function as receptors for circulating glucose levels, and contribute to glycemic counterregulatory responses. Using a thin-slice preparation that retains the structure of the carotid body and ⇑ Corresponding author. Fax: +52 3123161129x47408. E-mail addresses: [email protected], [email protected] (E. de AlvarezBuylla). 1089-8603/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2011.09.003

preserves the response of the glomus cells to hypoxia, it has been shown that low glucose levels and hypoxia converge in these cells to raise cytosolic calcium concentration to release transmitters, especially dopamine which, in turn, stimulates afferent sensory fibers [9]. Furthermore, decreased or increased plasma glucose concentration in the carotid sinus elicits a rapid neuroendocrine response to correct glycemic levels [10]. Local NaCN carotid chemoreceptor stimulation increases cephalic glucose retention, as well as induces peripheral hyperglycemia by sympathoadrenal activation [11] to counteract hypoglycemia [12]. Cardiorespiratory and glycemic responses are mediated through selective activation of glomus cells in the carotid body whose afferent pathways reach the solitary tract nucleus (STn) and periaqueductal gray [13,1]. Neither the mechanisms responsible for the glucose sensor activity in the carotid body, nor the central neural substrates involved in glucose regulation, have been precisely identified. Nitric oxide (NO) is an important mediator of a wide variety of functions in the CNS and peripheral organs, including neurotransmission and glucose metabolism [14,15]. Nitric oxide synthases and their mRNA are present in the ventromedial hypothalamic (VMH) neurons [16], where NO modulates glucose metabolism in

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astrocytes and neurons [17]. The presence of NO synthase isoforms and NO in the mitochondria also suggests that this molecule is important for glucose metabolism [18]. We have previously shown that intracisternal NO donors increase hyperglycemic response and brain glucose retention during normoxia in anesthetized rats; in contrast, during hypoxia, central NO administration inhibits these glucose responses [19]. Previous papers establish that NO within the STn is an important modulator of baroreceptor reflexes at this level, where carotid baroreceptor reflex controls heart rate in rats [20]. However, the role of NO in STn in response to chemoreceptor stimulation has not been investigated. We hypothesized that changes in NO within STn could modify the glycemic response initiated by carotid body stimulation. This study investigated whether changes in NO levels, by microinjection of an NO donor or an NOS inhibitor in STn, modifies brain glucose retention induced by local sodium cyanide (NaCN) carotid body chemoreceptors stimulated. Brain glucose retention was determined by arterial–venous (A–V) glucose differences measured in samples taken from the abdominal aorta and the jugular sinus [7,11] after an experimental maneuver. We also determined how gene expression of neuronal and inducible NO synthase isoforms (nNOS, iNOS) in STn was affected by carotid body stimulation in the presence or absence of nitroxidergic drugs in this nucleus. Methods Animals and general surgery Experiments were conducted in 83 male Wistar rats weighing 280–300 g. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals from National Institutes of Health [21]. Rats were fed standard rodent laboratory diet and water ad libitum and housed at 22–23 °C under a 12:12 light–dark cycle. Food was removed 12 h before surgery, but animals had free access to water containing 10% glucose; under these conditions glucose levels were stable during anesthesia. Buprenorphine (0.03 mg/ kg subcutaneously, Temgesic, Schering-Plough, Mexico) 5 min before surgical procedures was used as analgesic. Rats were anesthetized with intraperitoneal (i.p.) sodium pentobarbital (3.3 mg/100 g in saline solution, Aranda, Mexico) supplemented by a continuous i.p. infusion of the same anesthetic (0.063 mg/100 g/min in saline) monitored by wink and paw-pinch reflexes. Body temperature was kept at 37 ± 1 °C with a heating pad. Anesthetized animals were artificially ventilated through a tracheal cannula. Respiratory rate and tidal volume were based on pH, pO2 and pCO2 values in arterial blood obtained during experimental procedures, as well as 10 min before and at the end of experiment [22]. Permanent silastic catheters filled with heparin (1000 U/mL) were inserted into the abdominal aorta (accessed from the femoral artery) and jugular sinus (accessed from the right external jugular vein) without interrupting circulation in these vessels [7]. The correct placement of catheters was verified at the end of each experiment. Drugs and drug application The following drugs were used: (a) sodium cyanide (NaCN) (Fluka Biochemika, Sigma, Mexico) at a dose of 5 lg/100 g (diluted in 100 nL of freshly prepared sterile saline) [7]; (b) artificial cerebrospinal fluid (aCSF – 100 nL, freshly prepared and containing NaCl 145 mM, KCl 2.7 mM, MgCl 1.0 mM, CaCl2 1.2 mM, ascorbate 2.0 mM, NaH2PO4 2 mM, pH 7.3–7.4 [23]; (c) sodium nitroprusside (SNP; Sigma St. Louis, MO, USA) as an NO donor, at a dose of 300 lg (diluted in 100 nL of freshly prepared aCSF) [24]; (d) Nx-nitro-Larginine methyl ester (L-NAME; Sigma St. Louis, MO, USA) at a dose

of 250 lg (diluted in 100 nL of freshly prepared aCSF) [25] as NOS inhibitor. These drugs were diluted immediately before application to the final concentrations indicated. In sham experiments, the same volumes of aCSF or saline were injected. Carotid body receptor stimulation Carotid body receptor stimulation was performed as previously described [7]. Briefly, 5 lg/100 g NaCN in 100 lL saline/2 s was injected into the local circulation of the left carotid sinus, avoiding baroreceptor stimulation. NaCN was used as a carotid body chemoreceptor stimulator since its effects are equivalent to those observed in anoxemic anoxia [26]. As our intention was locally stimulate just one carotid sinus of the rat with a micro-dose of NaCN, the left carotid sinus was temporarily isolated from the cephalic circulation, while the right carotid sinus and aortic bodies were denervated (both aortic nerves and the right carotid sinus nerves were sectioned) (Fig. 1A). With this technique only the left carotid sinus is exposed to NaCN and within 15–16 s, NaCN is cleared into a washing cannula. The brief interruption of carotid circulation during stimulation does not produce cerebral ischemia [27]. Previous experiments conducted on isolated chemoreceptor fibers showed that an injection of 5 lg/100 g of NaCN into the carotid body circulation elicits electrical activity before the first signs of respiratory changes are observed, and these are reversible [28]. The anoxia obtained with these doses of NaCN is within the physiological pO2 range for carotid chemoreceptor sensitivity [28]. NaCN administered as described above, but after carotid nerve section did not have any effect [7], indicating that the observed responses were due to local carotid glomus cells chemoreceptor stimulation. For control experiments, aCSF (100 nL) instead of nitroxidergic drugs was injected into the STn, and saline (100 lL/ s) was injected instead of NaCN into the left carotid sinus, under the same conditions described above. Microinjection of drugs into the solitary tract nucleus Injections into the STn were done using a stereotaxic frame (Stoelting Co., Wood Dale, IL, USA) and the following coordinates from bregma: AP = 12.7 mm, L = 1.45 mm, V = 7.7 mm; incisor bar 3.3 mm above zero point [29]. The surface of the brain was reached with a 1/32 inch burr hole and a glass micropipette (50–60 lm external tip diameter; Microcap capillaries, Drummond Scientific Co., Broomall, PA, USA) filled with the solution to be injected, was inserted into the left STn. The micropipette was connected to a 0.5 mL Hamilton microsyringe with polyethylene tubing (PE 20) for injections. Nitroxidergic drugs were delivered in 100 nL of artificial cerebrospinal fluid (aCSF) during 3 s approximately. The volume of each injection was determined by measuring the movement of the fluid meniscus within the microinjector pipette. In control experiments the micropipette was directed to the gigantocellular reticular nucleus (Gi) the coordinates in this case were AP = 12.7 mm, L = 1.45 mm, V = 9.2 mm [29]. Local concentrations of micro-injected substances were estimated by a diffusion simulation program [30]. Cannula placement, neuronal injury and extent of diffusion were evaluated histologically after injections of equivalent volume of methylene blue (see below). We estimated, 30–40 s after microinjection, a diffusion of 1.5 mm around the injection site reaching 15–20% of the STn volume [30]. Once the last blood sample was drawn, the correct positioning of the micropipette tip site was corroborated by injecting 50–100 nL of methylene blue (10%) through the same micropipette (n = 42). Anesthetized rats were decapitated, the brains were removed, immediately frozen, and sectioned at 40 lm in a cryostat (CM-1800, Leica Microsystems, Nussloch, Germany). Sections were stained with cresyl violet

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Fig. 2. Representative frontal sections (40 lm) of the brain stem. (A) Rats that received the injection of nitroxidergic drugs into the nucleus of the tractus solitarius (STn). (B) Rats that received the injection of nitroxidergic drugs outside of the STn, into the region of the gigantocellular reticular nucleus (Gi). The sites of injection are indicated by arrows. Sections were stained with cresyl violet.

Fig. 1. Diagram illustrating the experimental design and protocol. (A) Placement of catheters and ligatures to locally perfuse left carotid sinus in order to stimulate carotid body chemoreceptors with sodium cyanide (NaCN). (B, C). Experimental protocol. aCSF, artificial cerebrospinal fluid; B, brain removal; cca, common carotid artery; CBR, carotid body receptors; cs, circulatory isolated carotid sinus; csn, carotid sinus nerve; eca, external carotid artery; G, glucose determination; ica, internal carotid artery; js, jugular sinus; la, lingual artery; L-NAME, Nx-nitro-Larginine methyl ester; NaCN, sodium cyanide; ph, pharyngeal artery; Sal, saline; SNP, sodium nitroprusside; STn, solitary tract nucleus.

for histological verification of the microinjection site and tissue damage (Fig. 2A and B). Blood sampling and measurements Glucose levels in the abdominal aorta were indistinguishable from those in the carotid artery. We therefore collected blood samples via catheters inserted into the abdominal aorta for arterial plasma measurements and from the jugular sinus for venous measurements. Heparin solution was first aspirated and catheters were then flushed three times with blood. Blood samples (arterial and

venous) (G in Fig. 1B and C) were collected from each rat as follows: two basal samples at t = 10 min and 5 min (values were averaged to obtain a basal level at t = 7.5 min). Nitroxidergic drugs or aCSF (as control) were injected into the STn at t = 4 min, while carotid body receptor stimulation (NaCN injection) or saline injection into the local circulation of the isolated carotid sinus were done at t = 0 min. Four experimental samples were then collected at t = 5, 10, 20 and t = 30 min (Fig. 1B). To compensate for fluid loss, rats received 0.3 mL saline after each sample was taken (0.15 mL of arterial blood and 0.15 mL of venous blood). Thus, blood volume taken from cannulated vessels amounted to 1.8 mL, less than 8% of total circulating volume. In some experiments L-NAME or aCSF injections were made 20 min after carotid chemoreceptor stimulation which was done at t = 0 min. In these cases three experimental samples were drawn at t = 25, 30 and 40 min, after cyanide stimulation (Fig. 1C). Blood samples were centrifuged and plasma was kept chilled until assayed. Plasma glucose concentration in lmol/mL was determined by the glucoseoxidase method with a glucose analyzer (Beckman Autoanalyzer, Beckman Coulter, CA, USA). Brain glucose retention was calculated in lmol/mL from arterial–venous (A–V) glucose differences in abdominal aorta and jugular sinus blood, respectively (Table 1). Blood flow was not considered for glucose retention estimation

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Table 1 Effects of carotid chemoreceptor stimulation with NaCN (5 lg/100 g/100 lL saline) on arterial and venous plasma glucose concentrations 4 min after an infusion of aCSF, SNP or LNAME in the NTS. Sample (min) 7.5 5 10 20 30

aCSF-2

SNP-2

L-NAME-2

Arterial (lmol/mL)

Venous (lmol/mL)

Arterial (lmol/mL)

Venous (lmol/mL)

Arterial (lmol/mL)

Venous (lmol/mL)

6.02 ± 0.27 7.25 ± 0.26* 7.56 ± 0.25* 7.72 ± 0.32* 7.80 ± 0.36*

5.33 ± 0.32 6.38 ± 0.27* 6.18 ± 0.29* 6.24 ± 0.49* 6.44 ± 0.52*

6.37 ± 0.25 6.89 ± 0.31*,+ 7.26 ± 0.26*,+ 7.36 ± 0.35*,+ 8.04 ± 0.43*

5.74 ± 0.32 6.08 ± 0.34 6.62 ± 0.36* 6.66 ± 0.37* 7.29 ± 0.39*

5.51 ± 0.27 6.54 ± 0.39*,§ 7.31 ± 0.50*,§ 8.42 ± 0.59*,+,§ 7.87 ± 0.68*

4.80 ± 0.32 5.64 ± 0.27 6.12 ± 0.35* 6.77 ± 0.22* 6.47 ± 0.32*

Each value indicates the mean ± SE; aCSF, artificial cerebrospinal fluid; L-NAME, Nx-nitro-L-arginine methyl ester; SNP, sodium nitroprusside. * p < 0.05, compares with its basal value. + p < 0.05 compares with its corresponding value in aCSF-2 group. § p < 0.05, compares the corresponding values between SNP and L-NAME groups.

as it has been shown that it does not change significantly after carotid chemoreceptor stimulation [7]. However, to discard any possible change in blood pressure after NaCN injection into the local carotid sinus circulation, arterial blood pressure was measured in the femoral artery in two control experiments with a pressure transducer (Ohmeda Pte. Ltd., Singapore) connected to a Grass polygraph (Grass Instruments Co., Quincy, MA, USA); the results obtained in these experiments did not show significant changes [7]. Arterial gases (pO2, pCO2) and pH levels were determined in a gas analyzer (Micro 13; Instrumentation Laboratory, Lexington, MA, USA) in mm Hg and absolute units, respectively. Glucose, pO2, pCO2 and pH values were within the range of standard curves at all times. Isolation of total RNA and reverse transcription –Polymerase Chain Reaction analysis To determine de novo expression of STn iNOS and nNOS genes, anesthetized rats (n = 35) were decapitated and brains dissected under RNase-free conditions. A 3 mm thick coronal section was dissected in a frozen brain frame that was kept frozen under 70 °C. From this section a piece of tissue of 100 mg. corresponding to the micropipette entering point to the STn area was used to obtain total RNA by means of an Ultraspec II kit (Biotecx Laboratories, Houston, TX, USA) [31], RNA was quantified in a spectrophotometer (Biophotometer; Eppendorf, Hamburg, Germany) and verified in agarose gels. RNA (1 lg) was used for reverse transcription (RT) and the cDNA obtained was amplified by a polymerase chain reaction (PCR) in the same tube with an EZ rTth RNA PCR Kit (Applied Biosystems, Branchburg, NJ, USA). For amplification (25 lL total volume), 1 lM of specific primers for iNOS [32] and nNOS [33] were added along with 300 lM of deoxynucleotide triphosphates, 2.5 mM Mn(OAc)2, 1X buffer for RT-PCR containing bicine (250 mM), potassium acetate (575 mM), 40% (w/v) glycerol, and 2.5 U of rTth DNA polymerase. As an internal control, 0.225 lM primers for specific amplification of the control RNA provided in the same RT–PCR kit were added to each reaction tube along with 1 lL of control RNA (pAW109, with concentration equivalent to 106 copies per mL of RNA, as described by the manufacturer). The same reaction condition used for the problem gene was used for the reference gene b-actin, adding 1 lM of specific primers. Mixtures were first incubated for 30 min at 60 °C for RT followed by 40 amplification cycles. Each cycle consisted of a 1min denaturing step at 94 °C, a 1-min aligning step at 60 °C, and a 1-min polymerizing step at 72 °C (MJ Research thermocycler, CA, USA). The RT-PCR product was visualized in 6% non-denaturing polyacrylamide gels (29:1) run for 2 h at 250 V followed by staining with silver nitrate [34]. Band density was measured in a Kodak densitometer and the relative quantification of the target gene was determined by calculating the ratio of the target gene to the house-

keeping gene b-actin. All gene expression analysis procedures were performed in rats at 40 min (B in Fig. 1B). Experimental protocol and number of animals in each experiment Animals were allowed to stabilize for at least 30 min after surgery, at which time they were randomized into the following groups: (a) aCSF-1 rats, with aCSF (100 nL into the STn) followed by saline (100 lL infused into the isolated carotid sinus), n = 7 for confirmation of STn injection, n = 7 for RT-PCR; (b) aCSF-2 rats, with aCSF (100 nL into the STn) followed by NaCN (5 lg/100 g in 100 lL saline infused into the isolated carotid sinus), n = 5 for identification of STn injection, n = 6 for RT-PCR; (c) SNP-1 rats (SNP dissolved in 100 nL aCSF into the STn) followed by saline (100 lL infused into the isolated carotid sinus), n = 10 for identification of STn injection, n = 6 for RT-PCR; (d) SNP-2 rats (SNP dissolved in 100 nL aCSF into the STn) followed by NaCN (5 lg/100 g in 100 lL saline infused into the isolated carotid sinus), n = 7 for identification of STn injection, n = 6 for RT-PCR; (e) L-NAME-1 rats (LNAME dissolved in 100 nL aCSF into the STn) followed by saline (100 lL infused into the isolated carotid sinus), n = 8 for identification of STn injection, n = 6 for RT-PCR; (f) L-NAME-2 rats (L-NAME dissolved in 100 nL aCSF into the STn) followed by NaCN (5 lg/ 100 g in 100 lL saline. infused into the isolated carotid sinus), n = 5 for identification of STn injection, n = 4 for RT-PCR (Fig. 1A and B); (g) aCSF-20 min rats (aCSF 100 nL into the STn) 20 min after NaCN (5 lg/100 g in 100 lL saline infused into the isolated carotid sinus), n = 3; (h) L-NAME-20 min rats (L-NAME dissolved in 100 nL into the STn) 20 min after NaCN (5 lg/100 g in 100 lL saline infused into the isolated carotid sinus), n = 3 (Fig. 1C). Data analysis Values are expressed as means ± SEM. All data were analyzed using the SPSS 12.0 software run on a PC computer for statistical significance by applying analysis of variance for a multiple comparisons test and Scheffé test to compare the data between groups. Significance was set at p < 0.05. For semi-quantitative target gene expression by RT-PCR, the ratio of the target gene to the housekeeping gene b-actin was used as arbitrary units that were standardized and a one-sample t-test was carried out. When basal glucose levels or basal brain glucose retention (mean value between t = 10 and 5 min) were compared with other values, significance is represented by an asterisk (⁄); the plus sign (+) was used to compare the significance between aCSF-2 group and SNP-2 or L-NAME-2 groups; a section sign (§) was used to compare the significance between SNP-2 and L-NAME-2 groups. When the number of mRNA levels for iNOS and nNOS were compared, the significance is represented as follows: The asterisk sign (⁄) compared aCSF-1 with aCSF-2, SNP-1 with SNP-2 or L-NAME-1 with

M. Lemus et al. / Nitric Oxide 25 (2011) 387–395

The plus sign (+) represents the comparison between aCSF-1 or aCSF-2 with their corresponding SNP or L-NAME groups. The comparison between both SNP and L-NAME groups was represented by the section sign (§).

L-NAME-2.

Results In our experiments the actual basal arterial blood glucose values were: in aCSF-1 and aCSF-2 rats, 6.39 ± 0.44 lmol/mL; in SNP-1 and SNP-2 rats, 6.09 ± 0.32 lmol/mL; in L-NAME-1 and L-NAME-2, 6.05 ± 0.30 lmol/mL; in aCSF, 20 min, 6.24 ± 0.23 lmol/mL and in L-NAME, 20 min, 6.68 ± 0.23 lmol/mL. Carotid chemoreceptor stimulation In anesthetized-rats (aCSF-1 rats), aCSF (100 nL into the STn) followed by saline (100 lL infused into the local circulation of the carotid sinus) did not result in significant changes in brain glucose retention at any of the times studied after saline injection (n = 7) (Fig. 3A). Brain glucose retention varied from 0.53 ± 0.06 lmol/mL at basal time to 0.44 ± 0.06 lmol/mL (p = 0.199) at t = 5 min, 0.53 ± 0.06 lmol/mL (p = 0.858) at t = 10 min, 0.46 ± 0.08 mol/mL

2.0

A

CBR

STn aCSF SNP L-NAME

1.5

SAL (aCSF-1) SAL (SNP-1) SAL (L-NAME-1)

1.0

Brain glucose retention (µmol/mL)

0.5

0.0

2.0

-10

-5

5

10

B

15

20

25

30

+§ STn

1.5

0

§

CBR

aCSF NaCN (aCSF-2) SNP NaCN (SNP-2) L-NAME NaCN (L-NAME-2)

*

*

*

§

*

*

* 1.0

0.5

0.0

391

(p = 0.448) at t = 20 min and 0.61 ± 0.10 lmol/mL (p = 0.472) at t = 30 min (Fig. 3A). This indicates that our anesthetized animal preparation is stable and that experimental injections into the carotid body or STn do not modify brain glucose retention. A second group of rats (aCSF-2) received aCSF into STn, but were injected with NaCN into the local circulation of the carotid sinus (carotid chemoreceptor stimulation). Under this local stimulation conditions NaCN only reaches the glomus cells and does not enter the systemic circulation. Nevertheless, this local stimulation induced a rapid increase in brain glucose retention (n = 5) similar to what we have observed before [7]. The levels rose from 0.69 ± 0.11 lmol/mL to 1.38 ± 0.23 lmol/mL at t = 10 min (p = 0.034) to 1.48 ± 0.23 lmol/mL at t = 20 min (p = 0.019) and 1.36 ± 0.23 lmol/mL (p = 0.019) at t = 30 min (Fig. 3B). This indicates that the mechanical effect of per-injection of aCSF into STn is not sufficient to alter the response associated with local carotid body stimulation with NaCN. It is important to note that under these conditions, arterial glucose concentration at 5, 10 and 20 min after the anoxic stimulus, significantly increased above the increase observed in venous glucose concentration (Table 1), that lead to observed changes in brain glucose retention. Carotid chemoreceptor stimulation after SNP infusion into solitary tract nucleus (STn) We next investigated the effects of NO increase in the STn before carotid body stimulation. NO donor, SNP, was infused into STn, before saline (SNP-1 rats, n = 10) or NaCN (SNP-2 rats, n = 7) injections into the local circulation of the carotid sinus. No significant changes in brain glucose retention were observed in the control SNP-1 rats that received saline into the carotid body. The brain glucose retention in SNP-1 rats varied between 0.36 and 0.47 lmol/mL (p = 0.391) (Fig. 3A). This indicates that SNP alone in STn did not modify brain glucose retention (Fig. 3A). Interestingly, however, SNP infusion into STn before injection of NaCN into the carotid body (SNP-2 group) blocked the increase in brain glucose retention normally induced by chemoreceptor stimulation. No significant differences were observed at any of the time points studied compared to basal levels (Fig. 3B); 0.63 ± 0.15 lmol/mL (basal), to 0.81 ± 0.16 lmol/mL (p = 0.239) at t = 5 min, 0.65 ± 0.21 lmol/mL (p = 0.932) at t = 10 min, 0.70 ± 0.17 lmol/mL (p = 0.648) at t = 20 min and 0.75 ± 0.15 lmol/mL (p = 0.424) at t = 30 min. In these experiments, the increase in arterial glucose concentrations after the anoxic stimulus was significantly lower when compared to aCSF-2 rats (p < 0.01) (Table 1). In control experiments a similar infusion of SNP outside the STn, in the region of the gigantocellular nucleus (Gi) (Fig. 2B), did not block the increase in brain glucose retention following chemoreceptor stimulation (results not shown) indicating that the effect of SNP were mediated through STn or its immediate vicinity. Carotid chemoreceptor stimulation after L-NAME infusion into solitary tract nucleus (STn)

-10

-5

0

5 10 15 20 Sample time (min)

25

30

Fig. 3. Brain glucose retention evoked by saline or carotid chemoreceptor stimulation (CBR) with NaCN, preceded by an infusion of aCSF, SNP or L-NAME into the solitary tract nucleus (STn). (A) Rats that received only saline infusion (100 nL) into the local circulation of the carotid sinus, preceded with the drugs as above. (B) Rats that received NaCN infusion (5 lg/100 g in 100 lL saline) into the local circulation of the carotid sinus preceded with the drugs as above. aCSF, artificial cerebrospinal fluid; L-NAME, Nx-nitro-L-arginine methyl ester; SAL, saline; SNP, sodium nitroprusside. Values are expressed as means ± standard error of the mean; comparisons between treatments over time were made by repeated measures using ANOVA and Scheffé post hoc tests; ⁄p < 0.05, compares with its basal value; +p < 0.05 compares with its corresponding value in aCSF-2 group; §p < 0.05, compares the corresponding values between SNP and L-NAME groups.

We next tested the effects of NOS inhibition in STn on brain glucose retention induced by carotid body stimulation. When the NOS inhibitor, L-NAME (250 lg diluted in 100 nL of aCSF) was infused into the STn and the carotid sinus was infused with saline (L-NAME-1, n = 8), no significant changes were observed in brain glucose retention (Fig. 3A). Brain glucose retention was similar to that of saline controls or when NO donor SNP was injected into STn. These results suggest that an inhibitor of NO also had no effect in the retention of glucose by the brain. In L-NAME-2 group, L-NAME was infused before carotid NaCN chemoreceptor stimulation (n = 5). In this case, brain glucose retention increased significantly for the entire duration of the experiment: from 0.70 ± 0.02 lmol/mL (basal

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value) to 0.90 ± 0.05 lmol/mL (p = 0.154) at t = 5 min, 1.19 ± 0.04 lmol/mL at t = 10 min (p = 0.004), 1.65 ± 0.14 lmol/mL at t = 20 min (p = 0.024) and 1.40 ± 0.06 lmol/mL at t = 30 min (p = 0.009) (Fig. 3B). Brain glucose retention reached higher levels in L-NAME-2 rats compared to those that were only exposed to the NaCN stimulus (aCSF-2 rats), reaching its highest value at 20 min after the NaCN stimulation. This difference, however, did not reach significance (p = 0.672) when compared to aCSF-2 rats. When arterial glucose levels in this group were compared to those in aCSF-2 group, a significant increase was observed at t = 20 min (p < 0.01) (Table 1). In contrast, comparison between brain glucose retentions in the L-NAME-2 group and the SNP-2 group yielded very significant differences at t = 10 min (p = 0.05), at t = 20 min (p = 0.005) and at t = 30 min (p = 0.012) (Fig. 3B). Carotid chemoreceptor stimulation before aCSF or L-NAME infusion into solitary tract nucleus (STn) To further analyze the results obtained in the previous section, in this group of experiments, the same dose of L-NAME (250 lg diluted in 100 nL of aCSF) was infused into the STn 20 min after the anoxic stimulus (n = 3). In this case, L-NAME infusion induced a rapid and significant increase, in brain glucose retention. When the values were compared with aCSF-20 min group (infusing aCSF instead of L-NAME). Brain glucose retention increased from 0.50 ± 0.09 lmol/mL (basal value) to 1.44 ± 0.16 at t = 25 min, 1.24 ± 0.21 lmol/mL at t = 30 min and 1.18 ± 0.03 lmol/mL at t = 40 min, although the results were only significant at t = 25 min (⁄p = 0.01) (Fig. 4B). These results indicated that an inhibitor of NOS had significant effects in the retention of glucose by the brain when the nitroxidergic drug in STn is accompanied by a carotid anoxic stimulation. Carotid chemoreceptor stimulation changes nNOS, but not iNOS gene expression in this nucleus We reasoned that endogenous levels of biosynthetic NO enzyme (nNOS and iNOS) could be modulated by NaCN stimulation and/or by NO donor SNP or NO inhibitor L-NAME. We first investi-

2.0

aCSF

Brain glucose retention (µmol/mL)

NaCN CBR

L-NAME

aCSF or L-NAME

*+

STn

1.5

*

* *

1.0

* 0.5

0.0

-10-7.5-5

0

20

25

30

35

40

Sample time (min) Fig. 4. Brain glucose retention evoked by carotid chemoreceptor stimulation with NaCN (5 lg/100 g in 100 lL saline), followed by an infusion of aCSF or L-NAME into the solitary tract nucleus (STn). Rats received an infusion of NaCN into the local circulation of the carotid sinus (CBR), followed at t = 20 min by aCSF (100 nL) or LNAME dissolved in aCSF 100 nL; aCSF, artificial cerebrospinal fluid; L-NAME, Nxnitro-L-arginine methyl ester; NaCN, sodium cyanide. Values are expressed as means ± standard error of the mean; ⁄p < 0.05, compares with its basal value; + p < 0.05 compares with its corresponding value in aCSF-2 group; §p < 0.05, compares the corresponding values between SNP and L-NAME groups.

gated if mRNA expression levels for nNOS and iNOS in STn were affected by carotid body stimulation. Basal relative levels of nNOS mRNA were always lower than those of iNOS (Fig. 4A and B). Interestingly, carotid body chemoreceptor stimulation after aCSF into STn (aCSF-2 rats, n = 6), more than doubled nNOS:b-actin ratio mRNA expression compared to non-stimulated aCSF-1 rats (0.31 ± 0.03 vs 0.71 ± 0.03) (p < 0.001) (Fig. 5). In contrast the mRNA expression ratio of iNOS:b-actin was not significantly different from controls (0.75 ± 0.06 in aCSF-1 rats and 0.77 ± 0.03 in aCSF-2 rats) (n = 7) (p = 0.98). This suggests that chemoreceptor stimulation results in a rapid increase in mRNA levels for nNOS in STn, but had not effect in STn levels of iNOS. We next investigated how the levels of nNOS and iNOS in STn were modulated by carotid body stimulation in the presence of nitroxidergic drugs in STn. In rats receiving SNP or L-NAME in STn prior to carotid body stimulation (SNP-2 and L-NAME-2 groups), carotid body stimulation resulted in an increase in nNOS similar to that observed for control rats micro-injected with aCSF in STn (aCSF-2 group) (Fig. 5A and B). In contrast, injections of SNP or L-NAME into STn, in the absence of carotid body stimulation, both increased by about 30% (0.75 ± 0.066 and 0.98 ± 0.035) the levels of iNOS in STn. In the presence of carotid body stimulation, values of iNOS returned to those present in control rats. These results indicate that carotid body stimulation is a major regulator of nNOS and that SNP and L-NAME did not significantly modify this response. However, iNOS appears not to be affected by carotid body stimulation, but it is slightly upregulated in the presence of donor or inhibitor of NO, only when the carotid body has not been stimulated.

Discussion In this study we evaluated the role of nitroxidergic drugs infused in the STn on brain glucose retention induced by a local anoxic stimulation to the carotid chemoreceptors. We showed in anesthetized rats that a brief, transient anoxic stimulus locally applied to the carotid sinus induced an increase in glucose retention by the brain (Figs. 3B and 4A), mostly due to a higher increase in arterial glucose concentration in comparison to a smaller venous glucose increase (Table 1). Interestingly, when an NO donor SNP was micro-injected in STn, prior to carotid body stimulation, completely blocked the increase in brain glucose retention normally induced by this chemoreceptor activation. In contrast, blocking NO synthase (NOS) with L-NAME, significantly increased brain glucose retention, but in these rats, the increased values of glucose retention did not reach significant differences when compared to its controls (aCSF-2 rats). Note that the injection of these nitroxidergic drugs did not induce, on their own, an effect in brain glucose retention. SNP also had no effect when injected outside of STn, in neighboring regions of the brainstem, in the Gi nucleus (Fig. 2B) which is also a glucose insensitive area [35]. These results confirm the important role that carotid body chemoreceptors play in glucose regulation [7,36,19], and show that this response is mediated through STn [37]. Moreover the work suggested that increased NO within STn serves to block the brain glucose retention initiated by the stimulation of the carotid body chemoreceptors. What could be the functional significance of this blockage? We infer that following carotid body receptors stimulation, levels of NO may increase in STn and this may help down to regulate the cascade of events that results in increased brain glucose retention. NO inhibits N-channel gating through cGMP and PKG with a decrease in Ca2 + influx through these channels [38]. How this affects different neuronal populations in STn and derives in an inhibitory effect in brain glucose retention initiated by chemoreceptor stimulation remains unknown. The principles

M. Lemus et al. / Nitric Oxide 25 (2011) 387–395

A

aCSF-1 aCSF-2

1.0

*

0.8 0.6 0.4 0.2 0.0

nNOS

iNOS

B +

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SNP-1 SNP-2

*

NOS-β -actin ratio

0.8 0.6 0.4 0.2 0.0

nNOS

iNOS

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1.0

*

0.8

*

L-NAME-1 L-NAME-2

§

0.6 0.4 0.2

+§

0.0

nNOS

iNOS

Fig. 5. Densitometric analysis of mRNA-nNOS and mRNA-iNOS in the STn, in the different groups studied. The samples were taken at t = 40 min after the injections into the carotid sinus. (A) aCSF-1 (aCSF + saline) and aCSF-2 (aCSF + NaCN) groups. (B) SNP-1 (SNP + saline) and SNP-2 (SNP + NaCN) groups. (C) L-NAME-1 (LNAME + saline) and L-NAME-2 (L-NAME + NaCN) groups. aCSF, artificial cerebrospinal fluid; L-NAME, Nx-nitro-L-arginine methyl ester; SNP, sodium nitroprusside. Data are means of relative density ± standard error of the mean; comparisons between treatments over time were made by repeated measures ANOVA using the Scheffé post hoc test. ⁄p < 0.05 vs corresponding non-stimulated group; +p < 0.05 vs aCSF corresponding groups; §p < 0.05 when SNP vs L-NAME groups were compared.

underlying the coupling of metabolic processes with neuronal activity and specially the role of neuron-glia metabolic interactions are still a subject of active debate. During the neuronal activity, a significant portion of glucose is taken by astrocytes while oxygen is mostly used within the neuronal population [39]. There is an activity-regulated transfer of glucose-derived metabolites from

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astrocytes to neurons [40]. Of course, the possibility that NO is involved in this shuttle cannot be excluded. NO-mediated inhibition of cytochrome c-oxidase may be a step of signaling process involved in the regulation of cellular functions in most cells, including astrocytes [41]. Following inhibition of mitochondrial respiration, glycolysis and glycogenolysis are essential for astrocytic responses to increasing energy demand [41,42], glycogen utilization can be interpreted as an increase in the effective astrocytic fraction of the total glucose used to feed ATP synthesis, and NO could modulate the balance between glucose consumption through the glycolytic pathway and the pentose phosphate pathway in neurons [41], as it seems to occur in the present study. Blockage of NO with L-NAME, 20 min after carotid chemoreceptor stimulation, significantly increased brain glucose retention response at t = 25 min after the stimulus when compared with control aCSF-20 min rats (Fig. 4), due to a significant increase in arterial glucose concentration 20 min after the local NaCN (Table 1). It is interesting to note that nNOS inhibition with L-NAME blocked the inhibitory action of NO. The possibility that nNOS response is not directly linked to the stimulus, but to the response is also important; i.e. the resulting increase in brain glucose retention, including the brainstem, could induce an increased NO in STn, and this could serve as a negative feedback to halt additional increase in brain glucose retention initiated at the carotid body, and channeled through STn. It is possible that these inhibitory effects are mediated through GABA in STn as NO induces an increase in GABAergic inhibitory synaptic potentials in the STn [43]. During baroreceptor activation, nNOS is upregulated in a similar way in STn [1,20]. It was recently showed that nNOS activation during hypoglycemia, stimulates NO production in the VMH glucose-inhibited neurons, necessary for the generation of the counterregulatory responses [44]. However, over-expressing nNOS or injecting NO donors into the VMH of nNOS knockout mice would not mimic physiological NO production [44]. Intracisternal infusion of an NO donor nitroglycerine, under normoxic conditions, increases brain glucose retention, while during the hypoxic state, a central NO donor does not enhance brain glucose retention [19]. Our data obtained in a whole animal, could be compared with previous observations using in vitro preparations, which suggest a dual effect of NO depending on PO2 conditions. NO donors inhibit chemosensory discharges during hypoxia, probably due to a vasodilatation effect that increases pO2 in the carotid body; and the same donor has the opposite effect during normoxia because of NO impairment of cytochrome oxidase redox activity [45]. The fact that unilateral SNP infusion into the STn, in the absence of an anoxic stimulation (SNP-1 rats), did not change nNOS mRNA expression indicates that NO in these experiments has a permissive role only when the anoxic stimulus is present (Fig. 5B). Before the anoxic stimulation, with the exception of L-NAME that induced a small but significant decrease in nNOS expression, the rats did not show changes neither in brain glucose retention nor in nNOS gene expression in the STn (Figs. 3A and 5A–C). In contrast, we demonstrated that carotid body stimulation induces a rapid increase in nNOS, one of the two biosynthetic enzymes involved in the production of NO, analyzed here. The anoxic stimulus activated neurons in the STn, to induce nNOS gene expression determined by a semi-quantitative PCR method (Fig. 5A). An NO donor SNP applied into the STn immediately before carotid chemoreceptor stimulation in SNP-2 rats, enhanced nNOS mRNA expression (Fig. 5B) and blocked the increase in brain glucose retention observed after the anoxic stimulus in control rats (Fig. 3B), as was previously observed in fetal animals [46]. Blockade of endogenous NO production with L-NAME in the STn without carotid chemoreceptor stimulation (L-NAME-1 rats) decreased the relative quantification of nNOS mRNA in the STn when compared to aCSF-1

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and SNP-1 rats (Fig. 5). In L-NAME-2 rats the levels of nNOS mRNA also decreased when compared to SNP-2 rats. These data suggested that NO-dependent glucose uptake pathways in the STn are only relevant during carotid chemoreceptor stimulation, and they are consistent with the idea that carotid chemoreceptor stimulation results in activation of nitroxidergic signaling in this nucleus [19]. In this regard, it should be noted that NO also modulates baroreceptor afferents at STn level, where a suppression of NOS in the STn fails to affect the baroreceptor reflex by increasing heart rate range during afferent stimulation [20]. Our data support a role for nNOS, rather than iNOS, at the level of STn in modulating the glycemic responses after a local hypoxic stimulus to carotid chemoreceptors (Fig. 5B and C). It is interesting to note that iNOS mRNA expression did not change significantly after carotid chemoreceptor stimulation. This indicated that iNOS signaling in the STn by itself was not sufficient to modify brain glucose retention in the time studied. Although carotid chemoreceptor stimulation alone did not induce iNOS expression in the STn, both nitroxidergic drugs in STn produced differential effects in this NOS isoform. While these drugs increased iNOS expression without NaCN stimulation when compared to aCSF-1 rats, after the anoxic stimulus they induced small but significant decreases in iNOS–b-actin (Fig. 5). NO derived from nNOS, rather than iNOS, also regulates induction of barosensitive neurons in the STn [47]. Although we do not know which neurons within the STn respond to SNP or L-NAME application, it is clear that specific population of neurons in the dorsomedial medulla acts as the main integration center to modulate the afferent signals from the carotid receptors via NO neuronal circuits to control glucose homeostasis. This observation further highlights the role of NO at the level of STn in the control of energy homeostasis [48,49,37]. In conclusion, our study suggests that carotid chemoreceptor activation results in changes in the levels of NO in STn most likely derived from nNOS activation, and that NO blocks brain glucose retention initiated by this stimulus. Acknowledgments The authors are deeply indebted to Dr. Arturo Álvarez-Buylla (Eli and Edithe Center of Regeneration Medicine and Stem Cell Research at UCSF) for his invaluable discussion and critical evaluation of the manuscript, and to Dr. Igor Pottosin from University of Colima for his art help. This work was supported by Grants: Fondo Ramón ÁlvarezBuylla de Aldana 330/05 and CONACYT P49376-Q. References [1] S.H.H. Chan, K.F. Chang, C.C. Ou, J.Y.H. Chan, Nitric oxide regulates c-fos expression in nucleus tractus solitarii induced by baroreceptor activation via cGMP-dependent protein kinase and cAMP response element-binding protein phosphorylation, Mol. Pharmacol. 65 (2004) 319–325. [2] P.E. Cryer, S.N. Davis, H. Shamoon, Hypoglycemia in diabetes, Diabetes Care 26 (2003) 1902–1912. [3] D.H. Wasserman, Four grams of glucose, Am. J. Physiol. Endocrinol. Metab. 296 (2009) E11–21. [4] S. Ritter, K. Bugarith, T.T. Dinh, Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation, J. Comp. Neurol. 432 (2001) 197–216. [5] E. Guillod-Maximin, A. Lorsignol, T. Alquier, L. Penicaud, Acute intracarotid glucose injection towards the brain induces specific c-fos activation in hypothalamic nuclei: involvement of astrocytes in cerebral glucose-sensing in rats, J. Neuroendocrinol. 16 (2005) 464–471. [6] A.L. Hevener, R.N. Bergman, C.M. Donovan, Hypoglycemic detection does not occur in the hepatic artery or liver. Findings consistent with a portal vein glucosensor locus, Diabetes 50 (2001) 399–403. [7] R. Alvarez-Buylla, E. Alvarez-Buylla, Carotid sinus receptors participate in glucose homeostasis, Respir. Physiol. 72 (1988) 347–360. [8] Y. Koyama, R.H. Cocker, E.E. Stone, D.B. Lacy, K. Jabbour, P.E. Williams, D.H. Wasserman, Evidence that carotid bodies play an important role in glucoregulation in vivo, Diabetes 49 (2000) 1434–1442.

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