Mary G. Garry

The mammalian exercise pressor reflex in health and disease

The exercise pressor reflex (a peripheral neural reflex originating in skeletal muscle) contributes significantly to the regulation of the cardiovascular system during exercise. Exercise‐induced signals that comprise the afferent arm of the reflex are generated by activation of mechanically (muscle mechanoreflex) and chemically sensitive (muscle metaboreflex) skeletal muscle receptors. Activation of these receptors and their associated afferent fibres reflexively adjusts sympathetic and parasympathetic nerve activity during exercise. In heart failure, the cardiovascular response to exercise is augmented. Owing to the peripheral skeletal myopathy that develops in heart failure (e.g. muscle atrophy, decreased peripheral blood flow, fibre‐type transformation and reduced oxidative capacity), the exercise pressor reflex has been implicated as a possible mechanism by which the cardiovascular response to physical activity is exaggerated in this disease. Accumulating evidence supports this conclusion. This review therefore focuses on the role of the exercise pressor reflex in regulating the cardiovascular system during exercise in both health and disease. Updates on our current understanding of the exercise pressor reflex neural pathway as well as experimental models used to study this reflex are presented. In addition, special emphasis is placed on the changes in exercise pressor reflex activity that develop in heart failure, including the contributions of the muscle mechanoreflex and metaboreflex to this pressor reflex dysfunction.

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freunds adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and/or GPR8.

Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation

In vitro superfusion was used to examine the effect of inflammation on the release of immunoreactive calcitonin gene-related peptide (iCGRP) and substance P(iSP) from the dorsal horn of rats. Three hours after carrageenan, hindpaws exhibited hyperalgesia, edema, and hyperthermia. Spontaneous and capsaicin-evoked release of iCGRP and iSP were significantly increased following inflammation. The enhanced release of iCGRP and iSP in the dorsal horn may serve as a biochemical marker for the development of hyperalgesia.

Neuroglobin, a novel member of the globin family, is expressed in focal regions of the brain

Hemoproteins are widely distributed among unicellular eukaryotes, plants, and animals. In addition to myoglobin and hemoglobin, a third hemoprotein, neuroglobin, has recently been isolated from vertebrate brain. Although the functional role of this novel member of the globin family remains unclear, neuroglobin contains a heme-binding domain and may participate in diverse processes such as oxygen transport, oxygen storage, nitric oxide detoxification, or modulation of terminal oxidase activity. In this study we utilized in situ hybridization (ISH) and RT-PCR analyses to examine the expression of neuroglobin in the normoxic and hypoxic murine brain. In the normoxic adult mouse, neuroglobin expression was observed in focal regions of the brain, including the lateral tegmental nuclei, the preoptic nucleus, amygdala, locus coeruleus, and nucleus of the solitary tract. Using ISH and RT-PCR techniques, no significant changes in neuroglobin expression in the adult murine brain was observed in response to chronic 10% oxygen. These results support the hypothesis that neuroglobin is a hemoprotein that is expressed in the brain and may have diverse functional roles.

Cardiogenic small molecules that enhance myocardial repair by stem cells

The clinical success of stem cell therapy for myocardial repair hinges on a better understanding of cardiac fate mechanisms. We have identified small molecules involved in cardiac fate by screening a chemical library for activators of the signature gene Nkx2.5, using a luciferase knockin bacterial artificial chromosome (BAC) in mouse P19CL6 pluripotent stem cells. We describe a family of sulfonyl-hydrazone (Shz) small molecules that can trigger cardiac mRNA and protein expression in a variety of embryonic and adult stem/progenitor cells, including human mobilized peripheral blood mononuclear cells (M-PBMCs). Small-molecule-enhanced M-PBMCs engrafted into the rat heart in proximity to an experimental injury improved cardiac function better than control cells. Recovery of cardiac function correlated with persistence of viable human cells, expressing human-specific cardiac mRNAs and proteins. Shz small molecules are promising starting points for drugs to promote myocardial repair/regeneration by activating cardiac differentiation in M-PBMCs.

Hypoxia-Inducible Factor-2α Transactivates Abcg2 and Promotes Cytoprotection in Cardiac Side Population Cells

Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic as well as oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. We observed that the cardiac SP cells increase in number more than 3-fold within 3 days of injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals increased expression of cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide and is associated with increased levels of &agr;-glutathione reductase protein expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene, we demonstrated that hypoxia-inducible factor (HIF)-2&agr; binds an evolutionary conserved HIF-2&agr; response element in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of Abcg2 expression by HIF-2&agr;. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2&agr; which functions with other factors to initiate a cytoprotective program for this progenitor SP cell population that resides in the adult heart.

Electrically induced static exercise elicits a pressor response in the decerebrate rat

1 The purpose of this investigation was to determine if activation of the exercise pressor reflex in the decerebrate rat induced circulatory responses comparable to those reported in large mammalian species. 2 To activate both mechanically and metabolically sensitive afferent fibres, static hindlimb contractions were induced by stimulating the cut ends of L4 and L5 spinal ventral roots in Sprague‐Dawley rats (300–400 g). To selectively stimulate mechanically sensitive receptors, hindlimb muscles were passively stretched. 3 In intact halothane‐anaesthetized animals (n= 10)), static contraction and passive stretch induced a decrease in mean arterial pressure (ΔMAP =−17 ± 3 and −8 ± 1 mmHg for contraction and stretch, respectively) and heart rate (HR). In contrast, MAP increased 23 ± 2 mmHg during contraction and 19 ± 3 mmHg during stretch in decerebrate rats (n= 10)). These pressor responses were accompanied by a significant tachycardia. In decerebrate animals, the reintroduction of halothane attenuated the increase in MAP and HR caused by both contraction and stretch. 4 In both anaesthetized and decerebrate rats, sectioning the spinal dorsal roots innervating the activated skeletal muscle eliminated responses to contraction and stretch. This finding indicated that an intramuscular neural reflex mediated the response to each stimulus. 5 The results demonstrate that a decerebrate preparation in the rat is a reliable model for the study of the exercise pressor reflex. Development of the model would enable the study of this reflex in a variety of pathological conditions and allow investigation of the mechanisms controlling cardiovascular responses to exercise in health and disease.

Mechanoreflex Mediates the Exaggerated Exercise Pressor Reflex in Heart Failure

Background— In heart failure, exercise elicits excessive increases in mean arterial pressure (MAP) and heart rate (HR). Using a novel rat model, we previously demonstrated that this exaggerated cardiovascular responsiveness is mediated by an overactive exercise pressor reflex (EPR). Although we previously determined that abnormalities in the group IV afferent neuron population (associated with the metabolic component of the reflex) initiate the development of the exaggerated EPR in heart failure, these fibers do not mediate the enhanced circulatory responses to exercise. Therefore, we hypothesized that the augmentation in EPR activity is primarily mediated by the mechanically sensitive component of the reflex (mediated predominately by activation of group III afferent fibers). Methods and Results— Male Sprague-Dawley rats were divided into 3 groups: sham (control), dilated cardiomyopathic (DCM), and neonatal capsaicin-treated animals (NNCAP, group IV afferent fibers ablated). Activation of the EPR by electrically induced static muscle contraction of the hindlimb resulted in larger increases in MAP and HR in DCM and NNCAP compared with sham animals. In all groups, administration of gadolinium (a selective blocker of mechanically sensitive receptors) within the hindlimb attenuated the MAP and HR responses to contraction. However, the magnitude of this reduction was greater in DCM and NNCAP compared with sham animals. Conclusions— From these data, we conclude that the muscle mechanoreflex mediates the exaggerated EPR that develops in heart failure. Moreover, these findings suggest that mechanoreflex overactivity in heart failure may be a compensatory response to functional alterations in group IV fibers. Given these findings, the muscle mechanoreflex may serve as a novel target in the treatment of the abnormal circulatory responses to exercise in heart failure.

Pharmacology of peripheral neuropeptide and inflammatory mediator release

Research conducted in the last 10 years has increased our knowledge on pain mechanisms substantially. Although many local tissue mediators, including neuropeptides, are known to exert pro-inflammatory effects, comparatively little is known about the actual tissue levels of these inflammatory mediators and their pharmacologic regulation. This article describes two new methods, clinical microdialysis and superfusion of dental pulp, which provide data on the pharmacology of peripheral neuropeptide and inflammatory mediator release. Collectively, these methods provide a biochemically based approach toward determining the mechanisms and management of orofacial pain.