Interactions among Endothelial Nitric Oxide Synthase, Cardiovascular System, and Nociception during Physiological and Pathophysiological States.

Nitric oxide synthase (NOS) plays important roles within the cardiovascular system in physiological states as well as in pathophysiologic and specific cardiovascular (CV) disease states, such as hypertension (HTN), arteriosclerosis, and cerebrovascular accidents. This review discusses the roles of the endothelial NOS (eNOS) and its effect on cardiovascular responses that are induced by nociceptive stimuli. The roles of eNOS enzyme in modulating CV functions while experiencing pain will be discussed. Nociception, otherwise known as the subjective experience of pain through sensory receptors, termed "nociceptors", can be stimulated by various external or internal stimuli. In turn, events of various cascade pathways implicating eNOS contribute to a plethora of pathophysiological responses to the noxious pain stimuli. Nociception pathways involve various regions of the brain and spinal cord, including the dorsolateral periaqueductal gray matter (PAG), rostral ventrolateral medulla (RVLM), caudal ventrolateral medulla, and intermediolateral column of the spinal cord. These pathways can interrelate in nociceptive responses to pain stimuli. The alterations in CV responses that affect GABAergic and glutamatergic pathways will be discussed in relation to mechanical and thermal (heat and cold) stimuli. Overall, this paper will discuss the aggregate recent and past data regarding pain pathways and the CV system.

A BRIEF SYNOPSIS OF MONOCLONAL ANTIBODY FOR THE TREATMENT OF VARIOUS GROUPS OF DISEASES.

Monoclonal antibodies (mAbs) are increasingly being prescribed to patients and investigated in the field of medicine and research. This class of medication is unique due to its ability to be engineered into targeting a specific receptor. Numerous studies and reviews have reported the efficacy, potency, and clinical usage of mAbs in the treatment of a variety of diseases ranging from autoimmune disorders to malignant cancers. However, very few publications classify and provide a brief synopsis of mAbs that includes their pharmacological profiles. mechanisms of action, uses, and side effects in a concise manner. Therefore, this review aims to classify the current mAbs drugs used in clinical practice according to system diseases by providing a brief summary for each of them. For example, regarding cardiovascular disorders, mAbs such as Abciximab, Bevacizumab, and Digoxin Immune Fab will be reviewed. Denosumab, used to treat musculoskeletal disorders, will be also discussed. In addition, mAbs such as Adalimumab, Eculizumab, Natalizumab used in autoimmune disorders and Alemtuzumab, Trastuzumab, Cetuximab, and Rituximab that are prescribed for tumors will be reviewed. Finally, we shall discuss two mAbs that are IL-6 antagonists, Tocilizumab and Siltuximab, which are in ongoing clinical trials as potential treatments of COVID-19. The mAbs have profound benefits against chronic and malignant conditions, and the overall purpose of this review is to illustrate the basic pharmacological profiles of mAbs that physicians may find useful in establishing their management protocols.

Role of neuronal nitric oxide synthase on cardiovascular functions in physiological and pathophysiological states.

This review describes and summarizes the role of neuronal nitric oxide synthase (nNOS) on the central nervous system, particularly on brain regions such as the ventrolateral medulla (VLM) and the periaqueductal gray matter (PAG), and on blood vessels and the heart that are involved in the regulation and control of the cardiovascular system (CVS). Furthermore, we shall also review the functional aspects of nNOS during several physiological, pathophysiological, and clinical conditions such as exercise, pain, cerebral vascular accidents or stroke and hypertension. For example, during stroke, a cascade of molecular, neurochemical, and cellular changes occur that affect the nervous system as elicited by generation of free radicals and nitric oxide (NO) from vulnerable neurons, peroxide formation, superoxides, apoptosis, and the differential activation of three isoforms of nitric oxide synthases (NOSs), and can exert profound effects on the CVS. Neuronal NOS is one of the three isoforms of NOSs, the others being endothelial (eNOS) and inducible (iNOS) enzymes. Neuronal NOS is a critical homeostatic component of the CVS and plays an important role in regulation of different systems and disease process including nociception. The functional and physiological roles of NO and nNOS are described at the beginning of this review. We also elaborate the structure, gene, domain, and regulation of the nNOS protein. Both inhibitory and excitatory role of nNOS on the sympathetic autonomic nervous system (SANS) and parasympathetic autonomic nervous system (PANS) as mediated via different neurotransmitters/signal transduction processes will be explored, particularly its effects on the CVS. Because the VLM plays a crucial function in cardiovascular homeostatic mechanisms, the neuroanatomy and cardiovascular regulation of the VLM will be discussed in conjunction with the actions of nNOS. Thereafter, we shall discuss the up-to-date developments that are related to the interaction between nNOS and cardiovascular diseases such as hypertension and stroke. Finally, we shall focus on the role of nNOS, particularly within the PAG in cardiovascular regulation and neurotransmission during different types of pain stimulus. Overall, this review focuses on our current understanding of the nNOS protein, and provides further insights on how nNOS modulates, regulates, and controls cardiovascular function during both physiological activity such as exercise, and pathophysiological conditions such as stroke and hypertension.

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla.

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.

Effects of medullary administration of a nitric oxide precursor on cardiovascular responses and neurotransmission during static exercise following ischemic stroke.

We have reported that in rats with a 90 min left middle cerebral artery occlusion (MCAO) and 24 h reperfusion, pressor responses during muscle contractions were attenuated, as were glutamate concentrations in the left rostral ventrolateral medulla (RVLM) and left caudal VLM (CVLM), but gamma-aminobutyric acid (GABA) levels increased in left RVLM and CVLM. This study determined the effects of L-arginine, a nitric oxide (NO) precursor, within the RVLM and (or) CVLM on cardiovascular activity and glutamate/GABA levels during static exercise in left-sided MCAO rats. Microdialysis of L-arginine into left RVLM had a greater attenuation of cardiovascular responses, a larger decrease in glutamate, and a significant increase in GABA levels during muscle contractions in stroke rats. Administration of N(G)-monomethyl-L-arginine, an NO-synthase inhibitor, reversed the effects. In contrast, L-arginine administration into left CVLM evoked a greater potentiation of cardiovascular responses, increased glutamate, and decreased GABA levels during contractions in stroked rats. However, L-arginine administration into both left RVLM and left CVLM elicited responses similar to its infusion into the left RVLM. These results suggest that NO within the RVLM and CVLM modulates cardiovascular responses and glutamate/GABA neurotransmission during static exercise following stroke, and that a RVLM-NO mechanism has a dominant effect in the medullary regulation of cardiovascular function.

Effects of inducible nitric oxide synthase blockade within the periaqueductal gray on cardiovascular responses during mechanical, heat, and cold nociception.

We have examined the role of inducible nitric oxide synthase (iNOS) within the dorsolateral periaqueductal gray mater (dlPAG) on cardiovascular responses during mechanical, thermal, and cold nociception in anesthetized rats. Mechanical stimulus was applied by a unilateral hindpaw pinch for 10 s that increased mean arterial pressure (MAP) and heart rate (HR). Bilateral microdialysis of a selective iNOS inhibitor, aminoguanidine (AGN; 10 µM), into the dlPAG for 30 min augmented MAP and HR responses during a mechanical stimulation. The cardiovascular responses recovered following discontinuation of the drug. Heat stimulus was generated by immersing one hindpaw metatarsus in a water bath at 52°C for 10 s, and this increased MAP and HR. Administration of AGN into the PAG potentiated these cardiovascular responses. Cardiovascular responses recovered following discontinuation of the drug. In contrast, application of a cold stimulus by immersing one hindpaw at 10°C for 10 s resulted in depressor and bradycardic responses. A second cold stimulus resulted in a response that was not significantly different from that prior to or after recovery from the AGN infusion. These results demonstrate that iNOS within the dlPAG plays a differential role in modulating cardiovascular responses during mechanical-, heat-, and cold-mediated nociception.

Transient middle cerebral artery occlusion and reperfusion alters inducible NOS expression within the ventrolateral medulla and modulates cardiovascular function during static exercise.

A major cause of stroke is cerebral ischemia in regions supplied by the middle cerebral artery (MCA). In this study, we hypothesized that compromised cardiovascular function during static exercise may involve altered expression of inducible NOS (iNOS) protein within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM). We compared cardiovascular responses and iNOS protein expression within the left and right sides of both RVLM and CVLM in sham-operated rats and in rats with a 90 min left-sided MCA occlusion (MCAO) followed by 24 h of reperfusion. Increases in blood pressure during a static muscle contraction were attenuated in MCAO rats compared with sham-operated rats. Also, iNOS expression within the left RVLM was augmented compared with the right RVLM in MCAO rats and compared with both RVLM quadrants in sham-operated rats. In contrast, compared with sham-operated rats and the right CVLM of MCAO rats, iNOS expression was attenuated in the left CVLM in left-sided MCAO rats. These data suggest that the attenuation of pressor responses during static exercise in MCAO rats involves overexpression of iNOS within the ipsilateral RVLM and attenuation in iNOS within the ipsilateral CVLM. Differential expression of iNOS within the medulla plays a role in mediating cardiovascular responses during static exercise following stroke.

Angiotensin II type-2 (AT2) receptor antagonism alters cardiovascular responses to static exercise and simultaneously changes glutamate/GABA levels within the ventrolateral medulla.

UNLABELLED: Angiotensin II receptors (Ang II), classified into AT1 and AT2 subtypes, are located in different regions of the central nervous system, including the cardiovascular control centers in the medulla oblongata. We previously reported the role of Ang II AT1 receptors within the medulla on cardiovascular responses and glutamate/GABA neurotransmission during the exercise pressor reflex [Patel, D., Böhlke, M., Phattanarudee, S., Kabadi, S., Maher, T.J., Ally, A., 2008. Cardiovascular responses and neurotransmitter changes during blockade of angiotensin II receptors within the ventrolateral medulla. Neurosci. Res. 60 (3), 340-348]. In this study, we investigated the role of the AT2 receptor subtype within the ventrolateral medullary region (VLM) in modulating increases in mean arterial pressure (MAP) and heart rate (HR) in response to static skeletal muscle contraction. METHODS: Using microdialysis methods in anesthetized rats, we administered AR-AT2 antagonists into the rostral (RVLM) and caudal (CVLM) VLM and determined its effects on cardiovascular responses and glutamate/GABA neurotransmission following muscle contraction. Bilateral microdialysis of a selective AT2 antagonist, PD 123319 (10 microM), for 30 min into the RVLM augmented MAP and HR responses during a static muscle contraction. Simultaneously, the drug increased glutamate and decreased GABA levels within the RVLM. After 60 min of discontinuation of the drug, only MAP and HR values but not the neurotransmitter levels in response to a muscle contraction returned to baseline. In contrast, bilateral microdialysis of the drug into the CVLM attenuated cardiovascular responses during a static muscle contraction, decreased glutamate and increased GABA. However, only the cardiovascular responses recovered after 60 min of discontinuation of the drug. These results demonstrate that AT2 within both RVLM and CVLM plays important differential roles in modulating neurotransmission and cardiovascular function during the exercise pressor reflex.

Effects of endothelial NOS antagonism within the periaqueductal gray on cardiovascular responses and neurotransmission during mechanical, heat, and cold nociception.

Nitric oxide (NO) is synthesized from L-arginine using NO synthase (NOS) enzyme that exists as 3 isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). We examined the role of eNOS within the dorsolateral periaqueductal gray mater (dlPAG) on cardiovascular responses along with glutamate and GABA concentrations during mechanical-, heat-, and cold-induced nociception in anesthetized rats. Mechanical stimulus was applied by a 10-second hindpaw pinch that increased mean arterial pressure (MAP) and heart rate (HR). Bilateral microdialysis of a selective eNOS antagonist, L-N(5)-(1-iminoethyl)ornithine (L-NIO; 10 microM), into the dlPAG had no effect on MAP or HR during a mechanical stimulation. Heat stimulus was generated by immersing a hindpaw metatarsus in a water-bath at 52 degrees C for 10 s which increased glutamate, GABA, MAP and HR. Administration of L-NIO into the dlPAG augmented cardiovascular responses and glutamate increase, but attenuated GABA changes during the heat stimulus. In contrast, application of a cold stimulus by immersing the hindpaw at 10 degrees C for 10 s resulted in decreases in MAP, HR, and glutamate. However, there was an increase in GABA concentration. Following microdialysis of L-NIO into the dlPAG, the responses to the cold stimulus was reversed i.e., the cold stimulus induced pressor and tachycardic responses, augmented glutamate, and attenuated GABA levels. These results demonstrate that eNOS within the dlPAG plays a differential role on the cardiovascular system during heat- and cold-mediated nociception via modulating glutamatergic/GABAergic neurotransmission. However, the mechanical stimulation had no effect on cardiovascular responses following eNOS antagonism within the dlPAG.

Endothelial NOS expression within the ventrolateral medulla can affect cardiovascular function during static exercise in stroked rats.

Temporary occlusion of the middle cerebral artery (MCA) causing damage to brain tissue occurs in the majority of human stroke victims. Reflex cardiovascular responses during static exercise were attenuated following transient MCA occlusion (MCAO) and reperfusion, mediated via alteration of the neuronal nitric oxide synthase (nNOS) protein isoform within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla (Ally, A., Nauli, S.M., Maher, T.J. 2005. Molecular changes in nNOS protein expression within the ventrolateral medulla following transient focal ischemia affect cardiovascular functions. Brain Res. [1055, 73-82]. We hypothesized that the endothelial NOS (eNOS) isoform within the RVLM and CVLM might also play a role in integrating cardiovascular function. Thus, we compared cardiovascular responses to static muscle contraction and eNOS expression within the four quadrants, i.e., left and right sides of both RVLM and CVLM in sham operated rats and in rats with a temporary 90-minute one-sided MCAO followed by 24 hour reperfusion. Increases in arterial pressure during a muscle contraction were attenuated in MCAO rats when compared to sham rats. Left-sided MCAO significantly decreased the expression of eNOS in the ipsilateral side but not contralateral RVLM, and to both RVLM quadrants in sham-operated rats. In contrast, compared to sham rats and the right CVLM quadrant of MCAO rats, eNOS expression was significantly increased in the left ipsilateral CVLM quadrant in left-sided MCAO rats. These data suggest that attenuation of cardiovascular responses during muscle contraction in MCAO rats may be partly due to a reduction in eNOS expression within the ipsilateral RVLM and an overexpression of eNOS within the ipsilateral CVLM. Results demonstrate that the eNOS protein within the medulla may play a significant role in mediating cardiovascular responses during static exercise in pathophysiological conditions, such as stroke.

Cardiovascular responses and neurotransmitter changes during blockade of angiotensin II receptors within the ventrolateral medulla.

Angiotensin II (Ang II) receptors are located in different regions of the brain, particularly within the cardiovascular control centers in the brainstem. These Ang II receptors are divided into AT1 and AT2 subtypes. We investigated the role of AT1 receptor subtype within the rostral (RVLM) and caudal (CVLM) ventrolateral medulla on cardiovascular responses and glutamate/GABA neurotransmission during static exercise using microdialysis in anesthetized rats. Bilateral microdialysis of a selective AT1 receptor antagonist, ZD7155 (10 microM), for 30 min into the RVLM attenuated increases in mean arterial pressure (MAP) and heart rate (HR) during a static muscle contraction. Glutamate concentrations within the RVLM decreased while GABA levels increased simultaneously during the contraction period when compared to those before ZD7155. After 60 min of discontinuation of ZD7155, MAP, HR, glutamate, and GABA levels in response to another muscle contraction returned to baseline levels. Conversely, bilateral microdialysis of ZD7155 into the CVLM potentiated cardiovascular responses during a static muscle contraction; glutamate concentrations increased while GABA levels within the CVLM decreased. All responses recovered after 60 min of discontinuation of ZD7155. These results demonstrate that medullary AT1 receptors play an important role in modulating both neurotransmission and cardiovascular function during static exercise.

Neuronal nitric oxide synthase (nNOS) blockade within the ventrolateral medulla differentially modulates cardiovascular responses and nNOS expression during static skeletal muscle contraction.

Nitric oxide (NO) is synthesized from L-arginine through the activity of the enzyme, NO synthase (NOS). Previous studies have demonstrated the role of the 3 isoforms of NOS, namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) in cardiovascular regulation. Local blockade of nNOS in RVLM vs. CVLM differentially alters local glutamate and GABA release, and thereby results in opposite cardiovascular responses to static muscle contraction (Brain Res. 2003, 977, 80-89). In this study, we examined whether nNOS antagonism within the RVLM and CVLM affected cardiovascular responses during the exercise pressor reflex and simultaneously modulated medullary nNOS protein expression using anesthetized rats. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (TRIM, 1.0 microM) for 120 min into the RVLM, potentiated cardiovascular responses during a static muscle contraction. Western blot analysis of nNOS expression within the RVLM showed significant attenuation of the protein when compared to the data obtained from control animals microdialyzed with vehicle. In contrast, bilateral application of TRIM into the CVLM attenuated cardiovascular responses during muscle contractions and increased nNOS protein expression within the CVLM. These results demonstrated that nNOS protein expression within the brainstem was pharmacologically altered by nNOS blockade within the RVLM or CVLM, which in turn might have contributed to the augmentation or attenuation of cardiovascular responses, respectively, during static exercise.

Modulation of cardiovascular responses and neurotransmission during peripheral nociception following nNOS antagonism within the periaqueductal gray.

Nitric oxide (NO) within the dorsal periaqueductal gray matter (dPAG) attenuated cardiovascular responses and changes in the concentrations of glutamate during both mechanical and thermal nociceptive stimulation [Ishide, T., Amer, A., Maher, T.J., Ally, A., 2005. Nitric oxide within periaqueductal gray modulates glutamatergic neurotransmission and cardiovascular responses during mechanical and thermal stimuli. Neurosci. Res. 51, 93-103]. Nitric oxide is synthesized from l-arginine via the enzyme, NO synthase (NOS), which exists in 3 isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). In this study, we examined the role of nNOS within the dPAG on cardiovascular responses and extracellular glutamate and GABA concentrations during mechanical and thermal nociception in anesthetized rats. The noxious mechanical stimulus was applied by a bilateral hindpaw pinch for 5 s that increased mean arterial pressure (MAP) and heart rate (HR) by 24+/-4 mm Hg and 41+/-7 bpm, respectively (n=10). Extracellular glutamate levels within the dPAG increased by 10.7+/-1.3 ng/mul while GABA concentrations decreased by 1.9+/-0.5 ng/microl. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (TRIM; 10.0 microM), into the dPAG had no effect on MAP, HR, glutamate and GABA values (P>0.05) during a mechanical stimulation. In a separate set of experiments, a noxious thermal stimulus was generated by immersing the metatarsus of a hindpaw in a water-bath at 52 degrees C for 5 s (n=10). Glutamate, MAP, and HR increased by 14.6+/-2 ng/microl, 45+/-6 mm Hg, and 47+/-7 bpm, while GABA decreased by 2.1+/-0.6 ng/microl. Administration of TRIM into the dPAG significantly enhanced the cardiovascular responses and glutamate increases (P<0.05) but further attenuated GABA changes (P<0.05) during subsequent thermal nociception. These results demonstrate that nNOS within the dPAG plays a differential role in modulating cardiovascular responses and glutamatergic/GABAergic neurotransmission during thermal and mechanical nociception.

Cardiovascular responses and neurotransmitter changes during static muscle contraction following blockade of inducible nitric oxide synthase (iNOS) within the ventrolateral medulla.

The enzyme nitric oxide synthase (NOS) which is necessary for the production of nitric oxide from L-arginine exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Our previous studies have demonstrated the roles of nNOS and eNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) in modulating cardiovascular responses during static skeletal muscle contraction via altering localized glutamate and GABA levels (Brain Res. 977 (2003) 80-89; Neuroscience Res. 52 (2005) 21-30). In this study, we investigated the role of iNOS within the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex. Bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 1.0 microM), for 60 min into the RVLM attenuated increases in mean arterial pressure (MAP), heart rate (HR), and extracellular glutamate levels during a static muscle contraction. Levels of GABA within the RVLM were increased. After 120 min of discontinuation of the drug, MAP and HR responses and glutamate/GABA concentrations recovered to baseline values during a subsequent muscle contraction. In contrast, bilateral application of AGN (1.0 microM) into CVLM potentiated cardiovascular responses and glutamate concentration while attenuating levels of GABA during a static muscle contraction. All values recovered after 120 min of discontinuation of the drug. These results demonstrate that iNOS within the ventrolateral medulla plays an important role in modulating cardiovascular responses and glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex.

Medullary monoamines and NMDA-receptor regulation of cardiovascular responses during peripheral nociceptive stimuli.

We have previously reported that AMPA-receptor blockade within the rostral ventrolateral medulla (RVLM) attenuates cardiovascular responses and extracellular concentrations of glutamate during mechanical, but not during thermal stimulation [Gray, T., Lewis III, E., Maher, T.J., Ally, A., 2001. AMPA-receptor blockade within the RVLM modulates cardiovascular responses via glutamate during peripheral stimuli. Pharmacol. Res. 43, 47-54]. In this study, we examined the role of NMDA-receptor blockade within the RVLM on cardiovascular responses and release of biogenic monoamines (serotonin [5HT], dopamine [DA], and norepinephrine [NE]) during both mechanical and thermal nociception using anesthetized Sprague-Dawley rats. Both mechanical and thermal stimulation have been shown to activate peripheral Adelta and C-fiber polymodal nociceptors. Noxious mechanical stimuli were induced by applying a pinch to alternate hindpaw for 5s while the noxious thermal stimuli involved immersion of the metatarsus of alternate hindpaw in a water bath at a temperature of 52 degrees C for 5 s. Mechanical stimulation increased mean arterial pressure (MAP), heart rate (HR), extracellular fluid 5HT, and DA concentrations (n=10). However, extracellular levels of NE were decreased within the RVLM. Furthermore, NMDA-receptor blockade with a competitive antagonist, AP-7 (200 nM), within the RVLM attenuated the cardiovascular responses and changes in 5HT and DA, but had no effect on NE levels. The thermal stimulation elicited similar increases in MAP and HR, however, extracellular levels of 5HT or DA did not change. Concentrations of NE were decreased during a thermal stimulation similar to the levels observed following mechanical stimuli. In contrast to mechanical stimuli, bilateral administration of AP-7 (200-1 mM) into the RVLM had no effect on cardiovascular responses, 5HT, DA or NE concentrations during a thermal stimulation. These results show that NMDA receptors within the RVLM most likely play a role in modulating cardiovascular responses by altering 5HT and DA concentrations within the RVLM during mechanical but not thermal nociception. Overall, the present study delineates the NMDA-receptor mediated central integrative mechanisms within the RVLM that coordinate processing of sensory impulses arising from peripheral noxious stimulation.

Molecular changes in nNOS protein expression within the ventrolateral medulla following transient focal ischemia affect cardiovascular functions.

The majority of human strokes involve an occlusion of the middle cerebral artery and subsequent damage to the brain tissues it perfuses. We have previously reported that reflex cardiovascular changes during a static muscle contraction are attenuated following transient middle cerebral artery occlusion (MCAO) and reperfusion [A. Ally, S.M. Nauli, T.J. Maher, Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion, Brain Res. 952 (2002) 176-187]. We hypothesized that the attenuation is a result of altered expression of neuronal nitric oxide synthase (nNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM). In this study, we have compared cardiovascular responses and nNOS protein expression within the four quadrants, i.e., left and right sides of both RVLM and CVLM in sham-operated rats (n = 10) and in rats with a temporary 90-min left-sided MCAO followed by 24 h reperfusion (n = 10). Increases in mean arterial pressure during a static muscle contraction were significantly attenuated in MCAO rats when compared to sham rats. The transient ischemia reduced nNOS expression within the ipsilateral RVLM quadrant compared to the contralateral RVLM or RVLM quadrants of control rats. In contrast, compared to sham rats and the right CVLM quadrant of MCAO rats, nNOS expression was significantly augmented in the ipsilateral CVLM in left-sided MCAO rats. These data suggest that the attenuation of cardiovascular responses during static muscle contraction in MCAO rats is partly due to a reduction in nNOS expression within the ipsilateral RVLM and an overexpression of nNOS abundance within the ipsilateral CVLM. Results demonstrate that nNOS expression within the medulla plays a significant role in mediating cardiovascular responses during static exercise in intact and pathophysiological conditions.

Neurochemistry within ventrolateral medulla and cardiovascular effects during static exercise following eNOS antagonism.

Nitric oxide synthase (NOS), necessary for the production of nitric oxide from l-arginine, exists in three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). We have previously demonstrated that blockade of nNOS within the rostral (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses to static exercise [Ishide, T., Nauli, S.M., Maher, T.J., Ally, A., 2003. Cardiovascular responses and neurotransmitter changes following blockade of nNOS within the ventrolateral medulla during static muscle contraction. Brain Res. 977, 80-89]. In this study, we have examined the effects of bilaterally microdialyzing a specific eNOS antagonist into the RVLM and CVLM on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex in anesthetized rats. Bilateral microdialysis of a selective eNOS antagonist, l-N(5)-(1-iminoethyl)ornithine (l-NIO; 10.0 microM) into the RVLM potentiated cardiovascular responses and increased extracellular fluid glutamate levels during a static muscle contraction. At the same time, levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered after discontinuation of the drug. In contrast, bilateral application of the eNOS antagonist into the CVLM attenuated cardiovascular responses and glutamate concentrations during a static muscle contraction, but augmented levels of GABA. These results demonstrate that eNOS within the ventrolateral medulla plays an important role in modulating glutamate/GABAergic neurotransmission, that in turn regulates the exercise pressor reflex. The present study provides further evidence of simultaneous sympathoexcitatory and sympathoinhibitory effects of nitric oxide within the RVLM and CVLM involved in the neural control of circulation during static exercise.

Nitric oxide within periaqueductal gray modulates glutamatergic neurotransmission and cardiovascular responses during mechanical and thermal stimuli.

We have previously reported that nitric oxide (NO) within the rostral ventrolateral medulla (RVLM) attenuates cardiovascular responses and extracellular concentrations of glutamate during thermal, but not during mechanical nociceptive stimulation (Ishide. T., Maher, T.J., Ally, A. 2003. Role of nitric oxide in the ventrolateral medulla on cardiovascular responses and glutamate neurotransmission during mechanical and thermal stimuli. Pharmacol. Res. 47, 59-68). In this study, we examined the role of nitric oxide within the dorsolateral periaqueductal gray matter (PAG), a higher center integrating nociceptive reflexes, on cardiovascular responses and glutamate release during both mechanical and thermal nociception using anesthetized Sprague-Dawley rats. Two types of stimuli were studied, both activating peripheral A(delta) and C fiber polymodal nociceptors. Noxious mechanical stimulus was given by applying a bilateral hindpaw pinch for 5 s. Mechanical stimulation of a hindlimb increased mean arterial pressure (MAP), heart rate (HR), and extracellular fluid glutamate within PAG by 20+/-3 mmHg, 37+/-6 bpm, and 1.7+/-0.3 ng/5 microl, respectively (n=10). Bilateral microdialysis of L-arginine (1.0 microM), a NO precursor, into the PAG significantly attenuated MAP, HR, and glutamate increases during a mechanical stimulation. Subsequent administration of N(G)-methyl-L-arginine (L-NMMA) (1.0 microM), a NO synthase inhibitor, into the PAG blocked the ability of NO within PAG to modulate the cardiovascular responses to mechanical stimulus. The noxious thermal stimulus was generated by immersing the metatarsus of a hindpaw in water-bath at a temperature of 52 degrees C for 5 s. Similar increases were observed following thermal stimulation: 35+/-5 mmHg, 40+/-6 bpm, and 1.14+/-0.4 ng/5 microl (n=10). L-Arginine attenuated both cardiovascular responses and glutamate increase during thermal nociception. These results demonstrate that NO within the dorsolateral PAG plays a role in modulating cardiovascular responses by altering glutamate concentrations during both thermal and mechanical nociception.

Cardiovascular responses and neurotransmitter changes following blockade of nNOS within the ventrolateral medulla during static muscle contraction.

Nitric oxide (NO) is synthesized from L-arginine through the activity of the synthetic enzyme, NO synthase (NOS). Previous studies have demonstrated the roles of the three isoforms of NOS, namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) in cardiovascular regulation. However, no investigation has been done to study their individual role in modulating cardiovascular responses during static skeletal muscle contraction. In this study, we determined the effects of microdialyzing a specific nNOS antagonist into the rostral (RVLM) and caudal ventrolateral medulla (CVLM) on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex using rats. We hypothesized that the NO modulation of the exercise pressor reflex was largely influenced by specific nNOS activity within the ventrolateral medulla. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (1.0 microM), for 30 or 60 min into the RVLM potentiated cardiovascular responses and glutamate release during a static muscle contraction. Levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered following discontinuation of the drug. In contrast, bilateral application of the nNOS antagonist into CVLM attenuated cardiovascular responses and glutamate release during a static muscle contraction, but augmented GABA release. These results demonstrate that nNOS in the ventrolateral medulla plays an important role in modulating glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex, and contributes to the sympathoexcitatory and sympathoinhibitory actions of NO within the RVLM and CVLM, respectively.