Chronic AT1 receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1 expression within the PVN in rats with heart failure

Allison C. Kleiber, Hong Zheng, Neeru M. Sharma, and Kaushik P. Patel

Am J Physiol Heart Circ Physiol. 2010 May;298(5):H1546-55. Epub 2010 Feb 19.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.

In my previous post, I summarized the article which discusses about how normalization of glutamatergic mechanisms in the paraventricular nucleus could be a possible mechanism by which exercise training (ExT) normalizes sympathetic outflow in heart failure (HF). As a follow up, I'm summarizing this article in which the authors investigated whether Ang II type 1 (AT1) receptors are involved in the normalization of PVN glutamatergic mechanisms. Studies by the same group have previously observed that ExT reduced the increased plasma Ang II levels associated with HF. Studies have shown that intravenous infusion of Ang II elevated the Fos immunoreactivity within the PVN, suggesting the possibility that PVN gene expression can be regulated by plasma Ang II levels. This let to the hypothesize that chronic AT1 receptor blockade in rats with HF would normalize PVN NMDA mediated renal sympathetic nerve activity (RSNA) responses and increased NR1 expression within the PVN. Chronic AT1 receptor blocker losartan was given at a dose of 50 mg/kg/day for a period of 3 weeks in drinking water. Three weeks of treatment with losartan normalized the NMDA induced (microinjected into the PVN) increase in RSNA.  In addition losartan treatment also normalized the elevated mRNA and protein expression of NMDA receptor subunit NR1. To address the question whether PVN received direct inputs from plasma ANG II since it cannot cross the blood brain barrier the authors suggested the possibility that the circumventricular organs such as subfornical organ (SFO) has direct projections to the PVN and it could have received signals from the SFO. The results from the present study suggest that normalization of plasma Ang II levels is one possible mechanism by which  exercise training normalizes enhanced gluatamatergic mechanisms associated with HF.

-Madhan

Chronic intermittent hypoxia augments sympatho-excitatory response to ATP but not to l-glutamate in the RVLM of rats

Daniel B. Zoccal, J. Pablo Huidobro-Toro b, Benedito H. Machado a, 

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil,

Nucleotide Research Lab, Department of Physiology, Faculty of Biological Sciences, P. Catholic University, Santiago, Chile 

Chemoreceptors located in the carotid bodies sense oxygen levels in the blood and are able to make respiratory and cardiovascular adjustments by activating neuronal mechanisms. When there is an increase in the activation of these chemoreceptors it can lead to the development of cardiovascular and respiratory pathological conditions. In this article, they were interested in obstructive sleep apnea (OSA), many of us already know that OSA can lead to the development of hypertension. Individuals that suffer from OSA experience chronic intermittent hypoxia (CIH is basically alteration between low and ambient air conditions). As we already know, glutamate is major neurotransmitter it has been shown that Glu release in the VLM cause excitation of pre-sympathetic and respiratory neurons. ATP may also play a role in this too. So the authors wanted to see whether CIH modulated the expression of Glutamatergic (Glu) and purinergic (ATP) receptors. They used microinjection and western blot techniques, in order to investigate their question. The investigators in this article were specifically interested in Bötzinger complex (Bötc) area. Bötc is important for respiratory control. When they were trying to functional identify this area using glutamate, they were looking for increase in sympathetic excitation along with a decrease in phrenic nerve activity.

For the microjection results, they found that there was no difference between the control and the CIH group in the response to glutamate. Then by western blot, they showed that there was no difference in the expression of Glutamatergic receptor protein between the groups. The glutamate receptors that they looked at using western blot were NMDAR1 and GluR2/3. For the purinergic receptors, they looked at using western blot were P2X1, P2X3, P2X4 and P2Y2. In the microinjection experiments, they found that there was a greater response to ATP in the CIH compared to the control suggesting that there is a possible up regulation of purinergic receptors due to CIH. The western blot results showed that there was increase in P2X3 and P2X4 protein in the CIH compared to control, which further supports their data. In CIH, there is increased SNA in these individuals. This article shows a possible factor that may contribute to the increase in SNA may be mediated by increased P2X3 and P2X4 in the VLM.

GABAergic mechanism in the rostral ventrolateral medulla contributes to the hypotension of moxonidine

Xin Ni1, Ding-Feng Su2, WeiWang4, Ming-Juan Xu5*, and Wei-ZhongWang1,6*

1Department of Physiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China; 2Department of Pharmacology, School of Pharmacy, Second Military MedicalUniversity, Shanghai 200433, China; 3Department of Neurobiology and Physiology, Ningxia Medical University, Yinchuan 750004, China; 4Department of Physiology, Nebraska MedicalCenter, Omaha 68198, USA; 5Department of Obstetrics and Gynecology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; and 6Key Laboratory of Molecula rNeurobiology, Ministry of Education, Second Military Medical University, Shanghai 200433, China Received 18 March 2010; revised 15 August 2010; accepted 1 September 2010; online publish-ahead-of-print 9 September 2010

Cardiovascular Research (2011) 89, 473–481 doi:10.1093/cvr/cvq289

This article investigated the effects of moxonidine, an antihypertensive drug on GABA _A  and GABA _B receptors along with GABA transmission in the RVLM. Mox activates  I₁R  that may mediate the release of GABA. So the investigators used several techniques, a couple that we currently use in the lab. They used microinjection, western blot and microdialysis. The major findings were that the effects of mox are mediated through the GABA receptors. They also demonstrated that there is an upregulation of GABA _A  and GABA _B  receptors and this was shown by western blot. By using microdialysis, they showed that there is an increase in the amount of GABA released in the RVLM after the use of mox.  These findings suggest that mox leads to an increase in GABAergic transmission in the RVLM that causes hypotension and an increase in sympathoinhibition.

GABAa α1 and α2 receptor subunit expression in rostral ventrolateral medulla in nonpregnant and pregnant rats

As most of you already know the RVLM contains the neurons responsible for excitatory input to preganglionic sympathetic neurons that produce vasoconstriction and increases in heart rate and contractility--- and you all definitely know that the neurotransmitter GABA inhibits sympathetic transmission in the RVLM. GABA_A receptors are heteroligomeric proteins that form ligand gated Cl^- channels. Expression of the different subunits can be regulated by numerous physiologic and pathophysiologic conditions. In this study the effects of pregnancy hormones (specifically estrogen and progesterone) on the expression of GABA_A subunits α_1-3. Foley et al. hypothesized that GABA_A receptors in the RVLM of late term pregnant rats may have higher expression of α₁GABA_A receptor subunit than the expression of α₂ or α₃ subunits.

Using PCR, quantitative PCR and immunoblots as their methods they set out to identify if there was indeed an effect by the progesterone metabolite 3α -OH-DHP (3α -hydroxy-dihydroprogesterone) on the α₁ receptor. The results showed that in the RVLM, the levels of GABA_A α₁ and α₂ receptor subunit mRNA and protein were similar between nonpregnant and late term pregnant rats, and the ration of GABA_A α₁ and α₂ receptor subunit mRNA expression was not different between groups. It seems that further studies have to be conducted on different GABA subunits that can be affected by neurosteroids.

The ventrolateral medulla and sympathetic regulation of arterial pressure.

ANN M. SCHREIHOFER AND ALAN F. SVED.  The ventrolateral medulla and sympathetic regulation of arterial pressure. In: Central regulation of autonomic functions.  2^nd Edition. Eds: I.J. Llewellyn-Smith and A.J. Verberne Oxford University Press, Inc. New York, 2011.

So in honor of our guest from Australia I thought it was timely to provide a posting on one of the chapters from Ida's recent book that will likely be considered the bible on neural control for at least the next 5-10 years.  The particular chapter I chose is of course most relevant to our studies on the RVLM, although other chapters in this book are also important in terms of our understanding of brainstem control of arterial pressure.  In this chapter Ann Schreihofer and Alan Sved provide a nice overview of the history and development of what the current knowledge is on brainstem control of sympathetic outflow.  They do a great job in discussing the number of techniques used to examine the ventrolateral medulla in term of it's anatomy and physiology.  Several nice figures combine immunohistochemistry, histology, BP and SNA responses etc. to illustrate the relationship between the CVLM and the RVLM.  There is also some clarification on the confusion caused by the original nomenclature of the C1 cells and that while serving as a useful marker for barosensitive, bulbospinal neurons controlling SNA, use glutamate not epinephrine serves as the primary neurotransmitters of RVLM neurons.  A thorough discussion of the roles of the RVLM and CVLM is provided.   There is a section of the caudal pressor area which while interesting, seems to pale in comparison to the importance of the CVLM and RVLM.  Finally, the last two sections are devoted to differential control and the role of the VLM in hypertension.  Both nice summaries but also highlight the number of unaswered questions that remain to be answered, some of which are being addressed in our laboratory.  Overall this is a chapter that every student, postdoc and faculty working in the field needs to be well-versed in so if you haven't done so recently or already, I would suggest giving it a look soon.

Posted by Pat

Hot off the press! Patterning of somatosympathetic reflexes reveals non-uniform organization of presympathetic drive from C1 and non-C1 RVLM neurons

It is well known that activation of muscle afferents by contracting muscle causes an increase in blood pressure. This is known as the somatopressor reflex. The reflex is driven by the SNS, and neurons in the RVLM mediate it. Some differential patterning of responses between different sympathetic nerves has been observed before, but not thoroughly investigated. This study investigates differential sympathetic nerve responses to graded activation of the afferent arm of the sciatic nerve, a.k.a. the somatosympathetic reflex. Furthermore, the relative contributions of C1 to non-C1 neurons in this response were examined.
Briefly, anesthetized rats were instrumeted to record arterial pressure and sympathetic activity from four nerves, cervical, splanchnic, lumbar, and renal. Stimulating electrodes were implanted on the left or right sciatic nerve. They performed sciatic nerve stimulation (ScNS) at varying intensities and amplitudes in normal rats. An additional set of experiments was performed in C1 neuron-depleted rats in which only splanchnic SNA was recorded.
Under normal conditions, ScNS produced double-peak responses in all but the cervical nerve. The peaks in the lumbar and renal nerves had slightly longer latency than splanchnic and cervical. In addition, the first peak of the splanchnic nerve was of larger amplitude than the second, but the opposite was seen in renal and lumbar nerves. In rats which had about 60% depletion of C1 neurons, the second peak in splanchnic SNA was abolished, but the first peak was only slightly attenuated.
These results suggest that, indeed, different sympathetic nerves have specific roles in the SNS response to muscle contraction. Moreover, C1 and non-C1 neurons are both involved in the response, each producing distinct peaks in splanchnic SNA, possibly according to the conduction velocity of their axons (C1 fast, non-C1 slow). This study provides even more evidence that SNA to specific vascular beds may be differentially controlled. It also highlights the importance of recording from multiple versus a single sympathetic output(s) in a single animal when performing in vivo experiments.
-Nick

Exercise training normalizes enhanced glutamate-mediated sympathetic activation from the PVN in heart failure

Kleiber AC, Zheng H, Schultz HD, Peuler JD, Patel KP.

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.

Am J Physiol Regul Integr Comp Physiol. 2008 Jun;294(6):R1863-72. Epub 2008 Apr 2.

 

Heart failure patients have high sympathetic outflow. Studies have shown that exercise training (ExT) normalizes muscle sympathetic nerve activity (MSNA), however the mechanisms are unknown. In this study the authors hypothesized the mechanisms by which ExT reduces MSNA is by normalizing the increased glutamatergic mechanisms within the PVN. Previous studies from the lab have shown that when PVN was injected with N-methyl-D-aspartic acid (NMDA) (a glutamate agonist that acts at the NMDA receptor), there was augmented renal sympathetic nerve activity (RSNA) in the heart failure rats compared to their normalized control. In this study the authors determined whether ExT reduces the RSNA response to NMDA in heart failure. Further they also determined whether ExT normalizes the expression of NMDA receptor subunit NR1 in heart failure rats. Heart failure was induced by left coronary artery ligation. Three weeks after inducing heart failure animals were given ExT using treadmills for 3 weeks. The animals ran for a period of 1hr/day at a speed of 20-25 m/min. RSNA and microinjection procedures were performed 6-8 wk after heart failure surgery or sham surgery. Microinjection of NMDA to hearf failure rats that are on ExT produced no changes in RSNA or MAP compared to control. This shows that ExT normalizes the augmented RSNA in response to NMDA microinjection in the PVN of heart failure rats. ExT also reduced the gene expression and protein levels of NMDA receptor subunit NR1 in the heart failure rats compared to heart failure rats that were not on ExT suggesting a possible mechanism by which RSNA responses to NMDA injected in to the PVN. These results suggest normalization of glutamatergic mechanisms is one possible way by which ExT normalizes sympathetic outflow in heart failure.

 

- Madhan

Local anaesthetics for acute reversible blockade of the sympathetic

Melissa M.J. Farnham, Paul M. Pilowsky

∗Australian School of Advanced Medicine, Level 1, Dow Corning Building, 3 Innovation Rd., Macquarie University, Macquarie, NSW 2109, Australia

Sinoaortic denervation (SAD) is a technique used to determine the role that the baroreceptors play in the control of blood pressure in different pathological states. In this article they discussed an alternative method to the current way of doing a SAD. They should how the use of lignocaine (10 – 30 min) and the bupivacaine (30 – 60 min) can yield result very similar to a complete (fig 2 and 3). The reversible SAD allows for more protocols can be done and also experiments can be done before and after the SAD. This new method allows for both intact and barodenervation to be investigated in the same animal this allows for greater statistical power and will reduce the number animals to be used for experiments. There are some disadvantages to this technique as well. Local anesthetics can have toxic effects on the cardiovascular system. This method will allow for more experiments in animals, along with increasing statistical power but another important reason to try this method that it reduces the amount of animals necessary for experiments thereby making this method more ethical.

Systemic Cholecystokinin Differentially Affects Baro-activated GABAergic

Susan C. Mobley, Daniel A. Mandel, and Ann M. Schreihofer
Department of Physiology, Medical College of Georgia, Augusta, Georgia

J Neurophysiol

First published August 16, 2006; doi:10.1152/jn.00526.2006


  In this article, the actions of Cholecystokinin (CCK) on CVLM and SNA were investigated. CCK will cause a decrease in splanchnic nerve activity (sSNA). CCK is released after the ingestion of a meal. First they looked how CCK will affect sSNA and arterial pressure (AP). They recorded the neurons activity in the CVLM during all procedures. what they found was that in the majority of animals depressor responses within the first 15 seconds. They also found that the response was not a smooth but that it decrease some at first and then came back up and was followed by a greater depressor response in AP and also in sSNA (see fig 1A). In a few animals there was no AP depressor response however, there was a decrease in the sSNA. Then the neurons were labeled with biotinamide and the GABAergic phenotype was confirmed.Next they investigated how CCK would infleunce the activity of baro-inhibited neurons. By using aortic snare AP was raised and those neurons that had decrease in activity were considered baro-inhibited CVLM neurons. Then CCK was injected and the activity of these neuron was decreased and several neurons were silenced. In order to determine if CVLM was essential to the effects of CCK, CVLM was inhibited and CCK was injected. Before the muscimol (mus), a GABA A receptor agonist, they gave phenylbiguanide (PBG) to show the maximal activity of CVLM, next CCK was given they saw a decrease in sSNA, AP and HR. Then they gave mus bilaterally and then injected CCK what they saw was an increase in SNA and a decrease in HR. As for the PBG after mus it was reversed also instead they saw increase in all responses. Because inhibiting the CVLM causes an increase in SNA it could be possible that CCK could be potentiating the affects of Acetylocholine at the nicotinic receptor. So they gave hydralazine a vasodilator to cause an increase in sSNA and then gave CCK which inhibited the sSNA. Therefore the increase seen in the muscimol protocol was not due to the elevation of SNA.

 The interesting thing is that PBG maximally activates CVLM neurons but CCK only activates about 30% of the CVLM neuron population suggesting that certain neurons are resposible for the control of specific neurons in RVLM that control specific nerves. for instance injecting CCK causes increase in lumbar SNA. CCK also has cardiovascular responses it mediate, it can cause vasodilation at the blood vessels. It can also cause bradycardia by acting at CCK A receptors on the heart, this is most likely why after inhibiting the CVLM bilaterally, there was still a decrease in HR. CCK therefore does not depend on the CVLM pathway to control HR. PBG however does which is why after the muscimol there was increases in HR, AP and sSNA.

 

Lateralisation of projections from the rostral ventrolateral medulla to sympathetic preganglionic neurons in the rat

Elizabeth A. Moon, Ann K. Goodchild, Paul M. Pilowsky
Brain Res. 2002 Mar 8;929(2):181-90.

It is well known that presymapthetic neurons in the RVLM is important in tonic and reflex control of blood pressure in the arteries and sympathetic nerve activity. In this article the authors were interested in identifying how the sympathoexcitatory neurons form connections with sympathetic preganglionic neurons (SPN). They tested this by three different approaches. 1) Retrograde tracers were used to label the SPN that project to the adrenal gland or the superior cervical ganglion (SCG). Antrograde tracers were used from the pressor sites in the RVLM in order to identify whether lateral projections from the bulbospinal neurons project to the SPN that innervates the SCG and adrenal gland. 2) To identify the degree of lateralization retrograde tracers were injected unilaterally in to the spinal cord. 3) Finally they used a electrophysiological approach to stimulate RVLM with glutamate and recorded the activity from adrenal and cervical sympathetic nerves in order to examine the functional aspect of lateralization.
The significant findings of the present study are anterograde and retrograde experiments showed that the presympathetic neurons from the RVLM that form connections with SPN that innervate the SCG are bilateral. Interestingly projections to the adernal gland are ipsilateral. The second study showed that projections from RVLM to both the upper and lower level of the thoracic spinal cord (intermediolateral cell column) are predominantly ipsilateral. Inspite of this differential anatomical lateralization, the final study with glutamate microinjection showed no difference in nerve response even when different sides of the RVLM were injected.

- Madhan

Effect of renal sympathetic denervation on glucose metabolism in patients with resistant hypertension: a pilot study.

As most of you probably know, a study came out a couple of years showing that renal sympathetic nerve denervation can dramatically lower blood pressure in a hypertensive patient.  While the reno-centric scientists out there said "See! I TOLD you it was the kidney!", the evidence as a whole suggested that a more global phenomenon was occurring, since whole body norepinephrine spillover and sympathetic activity to the muscle were reduced in these patients.  In addition to affecting blood pressure, symptahetic overactivity can negatively affect glucose metabolism.  When beta adrenergic receptors in the liver are stimulated by cuirculating epinephrine, liver cells increase glucose production and subsequently plasma glucose and inculin levels increase. In turn, insulin stimulates sympathetic nerve activity, forming a cycle of activation. The authors guessed that glucose metabolism would benefit from renal denervation as well.
They performed the procedure in 37 patients with resistant hypertension and assigned 13 to the control group. A catheter was inserted into the renal artery and radiofrequency pulses were used ot heat the tip of the catheter and ablate the artery at various spots. This procedure kills any nerves running in and around the artery and causes a scar to form that prevents reinnervation. Blood pressure and markers of glucose metabolism were measured before and 1 and 3 months after the study.
The authors found that renal denervation had a marked effect on glucose metabolism. It is important to note that the patients' glucose and insulin levels were only slightly above normal before the study. fasting gluicose decreased, baseline insulin levels decreased, and blood pressure decreased as expected. Interestingly, some members in the trestment group were 'cured' of diabetes or pre-diabetes, depending on how you look at it. Now, does this prove that denervation blunts central sympathetic outflow to the adrenal gland and thus epinephrine secretion to cause these effects? No. Is it enough evidence to be suggestive? To me, yes. But then again, the appearance of the moon may suggest that it is made of cheese.  In any case, this study reminds one to remember the BIG PICTURE. Sympathetic overactivity means much more than just blood pressure.

Link to article

-Nick