Bacterial growth dynamics in a rhythmic symbiosis.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for ∼6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage.

Consequences of PDGFRα+ fibroblast reduction in adult murine hearts.

Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation.

BACKGROUND: Cardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. METHODS AND RESULTS: We evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70-80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. CONCLUSIONS: These studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Sensory Afferent Renal Nerve Activated Gαi2 Subunit Proteins Mediate the Natriuretic, Sympathoinhibitory and Normotensive Responses to Peripheral Sodium Challenges.

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (µeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

Natriuresis During an Acute Intravenous Sodium Chloride Infusion in Conscious Sprague Dawley Rats Is Mediated by a Blood Pressure-Independent α1-Adrenoceptor-Mediated Mechanism.

The mechanisms that sense alterations in total body sodium content to facilitate sodium homeostasis in response to an acute sodium challenge that does not increase blood pressure have not been fully elucidated. We hypothesized that the renal sympathetic nerves are critical to mediate natriuresis via α1- or ß-adrenoceptors signal transduction pathways to maintain sodium balance in the face of acute increases in total body sodium content that do not activate the pressure-natriuresis mechanism. To address this hypothesis, we used acute bilateral renal denervation (RDNX), an anteroventral third ventricle (AV3V) lesion and α1- or ß-antagonism during an acute 1M NaCl sodium challenge in conscious male Sprague Dawley rats. An acute 1M NaCl infusion did not alter blood pressure and evoked profound natriuresis and sympathoinhibition. Acute bilateral RDNX attenuated the natriuretic and sympathoinhibitory responses evoked by a 1M NaCl infusion [peak natriuresis (µeq/min) sham 14.5 ± 1.3 vs. acute RDNX: 9.2 ± 1.4, p < 0.05; plasma NE (nmol/L) sham control: 44 ± 4 vs. sham 1M NaCl infusion 11 ± 2, p < 0.05; acute RDNX control: 42 ± 6 vs. acute RDNX 1M NaCl infusion 25 ± 3, p < 0.05]. In contrast, an AV3V lesion did not impact the cardiovascular, renal excretory or sympathoinhibitory responses to an acute 1M NaCl infusion. Acute i.v. α1-adrenoceptor antagonism with terazosin evoked a significant drop in baseline blood pressure and significantly attenuated the natriuretic response to a 1M NaCl load [peak natriuresis (µeq/min) saline 17.2 ± 1.4 vs. i.v. terazosin 7.8 ± 2.5, p < 0.05]. In contrast, acute ß-adrenoceptor antagonism with i.v. propranolol infusion did not impact the cardiovascular or renal excretory responses to an acute 1M NaCl infusion. Critically, the natriuretic response to an acute 1M NaCl infusion was significantly blunted in rats receiving a s.c. infusion of the α1-adrenoceptor antagonist terazosin at a dose that did not lower baseline blood pressure [peak natriuresis (µeq/min) sc saline: 18 ± 1 vs. sc terazosin 7 ± 2, p < 0.05]. Additionally, a s.c. infusion of the α1-adrenoceptor antagonist terazosin further attenuated the natriuretic response to a 1M NaCl infusion in acutely RDNX animals. Collectively these data indicate a specific role of a blood pressure-independent renal sympathetic nerve-dependent α1-adrenoceptor-mediated pathway in the natriuretic and sympathoinhibitory responses evoked by acute increases in total body sodium.

Hypothalamic Paraventricular Nucleus Gαi2 (Guanine Nucleotide-Binding Protein Alpha Inhibiting Activity Polypeptide 2) Protein-Mediated Neural Control of the Kidney and the Salt Sensitivity of Blood Pressure.

We have previously reported that in salt-resistant rat phenotypes brain, Gαi2 (guanine nucleotide-binding protein alpha inhibiting activity polypeptide 2) proteins are required to maintain blood pressure and sodium balance. However, the impact of hypothalamic paraventricular nucleus (PVN) Gαi2 proteins on the salt sensitivity of blood pressure is unknown. Here, by the bilateral PVN administration of a targeted Gαi2 oligodeoxynucleotide, we show that PVN-specific Gαi2 proteins are required to facilitate the full natriuretic response to an acute volume expansion (peak natriuresis [µeq/min] scrambled (SCR) oligodeoxynucleotide 41±3 versus Gαi2 oligodeoxynucleotide 18±4; P<0.05) via a renal nerve-dependent mechanism. Furthermore, in response to chronically elevated dietary sodium intake, PVN-specific Gαi2 proteins are essential to counter renal nerve-dependent salt-sensitive hypertension (mean arterial pressure [mm Hg] 8% NaCl; SCR oligodeoxynucleotide 128±2 versus Gαi2 oligodeoxynucleotide 147±3; P<0.05). This protective pathway involves activation of PVN Gαi2 signaling pathways, which mediate sympathoinhibition to the blood vessels and kidneys (renal norepinephrine [pg/mg] 8% NaCl; SCR oligodeoxynucleotide 375±39 versus Gαi2 oligodeoxynucleotide 850±27; P<0.05) and suppression of the activity of the sodium chloride cotransporter assessed as peak natriuresis to hydrochlorothiazide. Additionally, central oligodeoxynucleotide-mediated Gαi2 protein downregulation prevented PVN parvocellular neuron activation, assessed by FosB immunohistochemistry, in response to increased dietary salt intake. In our analysis of the UK BioBank data set, it was observed that 2 GNAI2 single nucleotide polymorphism (SNP) (rs2298952, P=0.041; rs4547694, P=0.017) significantly correlate with essential hypertension. Collectively, our data suggest that selective targeting and activation of PVN Gαi2 proteins is a novel therapeutic approach for the treatment of salt-sensitive hypertension.

Role of the afferent renal nerves in sodium homeostasis and blood pressure regulation in rats.

NEW FINDINGS: What is the central question of this study? What are the differential roles of the mechanosensitive and chemosensitive afferent renal nerves in the reno-renal reflex that promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to sodium homeostasis? What is the main finding and its importance? The mechanosensitive afferent renal nerves contribute to an acute natriuretic sympathoinhibitory reno-renal reflex that may be integrated within the paraventricular nucleus of the hypothalamus. Critically, the afferent renal nerves are required for the maintenance of salt resistance in Sprague-Dawley and Dahl salt-resistant rats and attenuate the development of Dahl salt-sensitive hypertension. ABSTRACT: These studies tested the hypothesis that in normotensive salt-resistant rat phenotypes the mechanosensitive afferent renal nerve (ARN) reno-renal reflex promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to fluid and electrolyte homeostasis. Selective ARN ablation was conducted prior to (1) an acute isotonic volume expansion (VE) or 1 m NaCl infusion in Sprague-Dawley (SD) rats and (2) chronic high salt intake in SD, Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. ARN responsiveness following high salt intake was assessed ex vivo in response to noradrenaline and sodium concentration (SD, DSR and DSS) and via in vivo manipulation of renal pelvic pressure and sodium concentration (SD and DSS). ARN ablation attenuated the natriuretic and sympathoinhibitory responses to an acute VE [peak natriuresis (µeq min-1 ) sham 52 ± 5 vs. ARN ablation 28 ± 3, P < 0.05], but not a hypertonic saline infusion in SD rats. High salt (HS) intake enhanced ARN reno-renal reflex-mediated natriuresis in response to direct increases in renal pelvic pressure (mechanoreceptor stimulus) in vivo and ARN responsiveness to noradrenaline ex vivo in SD, but not DSS, rats. In vivo and ex vivo ARN responsiveness to increased renal pelvic sodium concentration (chemoreceptor stimulus) was unaltered during HS intake. ARN ablation evoked sympathetically mediated salt-sensitive hypertension in SD rats [MAP (mmHg): sham normal salt 102 ± 2 vs. sham HS 104 ± 2 vs. ARN ablation normal salt 103 ± 2 vs. ARN ablation HS 121 ± 2, P < 0.05] and DSR rats and exacerbated DSS hypertension. The mechanosensitive ARNs mediate an acute sympathoinhibitory natriuretic reflex and counter the development of salt-sensitive hypertension.

Platelet-derived growth factor receptor-α is essential for cardiac fibroblast survival.

Platelet-derived growth factor receptor α (PDGFRα), a receptor tyrosine kinase required for cardiac fibroblast development, is uniquely expressed by fibroblasts in the adult heart. Despite the consensus that PDGFRα is expressed in adult cardiac fibroblasts, we know little about its function when these cells are at rest. Here, we demonstrate that loss of PDGFRα in cardiac fibroblasts resulted in a rapid reduction of resident fibroblasts. Furthermore, we observe that phosphatidylinositol 3-kinase signaling was required for PDGFRα-dependent fibroblast maintenance. Interestingly, this reduced number of fibroblasts was maintained long-term, suggesting that there is no homeostatic mechanism to monitor fibroblast numbers and restore hearts to wild-type levels. Although we did not observe any systolic functional changes in hearts with depleted fibroblasts, the basement membrane and microvasculature of these hearts were perturbed. Through in vitro analyses, we showed that PDGFRα signaling inhibition resulted in an increase in fibroblast cell death, and PDGFRα stimulation led to increased levels of the cell survival factor activating transcription factor 3. Our data reveal a unique role for PDGFRα signaling in fibroblast maintenance and illustrate that a 50% loss in cardiac fibroblasts does not result in lethality.NEW & NOTEWORTHY Platelet-derived growth factor receptor α (PDGFRα) is required in developing cardiac fibroblasts, but a functional role in adult, quiescent fibroblasts has not been identified. Here, we demonstrate that PDGFRα signaling is essential for cardiac fibroblast maintenance and that there are no homeostatic mechanisms to regulate fibroblast numbers in the heart. PDGFR signaling is generally considered mitogenic in fibroblasts, but these data suggest that this receptor may direct different cellular processes depending on the cell's maturation and activation status.

Resident fibroblast expansion during cardiac growth and remodeling.

Cardiac fibrosis, denoted by the deposition of extracellular matrix, manifests with a variety of diseases such as hypertension, diabetes, and myocardial infarction. Underlying this pathological extracellular matrix secretion is an expansion of fibroblasts. The mouse is now a common experimental model system for the study of cardiovascular remodeling and elucidation of fibroblast responses to cardiac growth and stress is vital for understanding disease processes. Here, using diverse but fibroblast specific markers, we report murine fibroblast distribution and proliferation in early postnatal, adult, and injured hearts. We find that perinatal fibroblasts and endothelial cells proliferate at similar rates. Furthermore, regardless of the injury model, fibroblast proliferation peaks within the first week after injury, a time window similar to the period of the inflammatory phase. In addition, fibroblast densities remain high weeks after the initial insult. These results provide detailed information regarding fibroblast distribution and proliferation in experimental methods of heart injury.

Tracking Adventitial Fibroblast Contribution to Disease: A Review of Current Methods to Identify Resident Fibroblasts.

Cells present in the adventitia, or outermost layer of the blood vessel, contribute to the progression of vascular diseases, such as atherosclerosis, hypertension, and aortic dissection. The adventitial fibroblast of the aorta is the prototypic perivascular fibroblast, but the adventitia is composed of multiple distinct cell populations. Therefore, methods for uniquely identifying the fibroblast are critical for a better understanding of how these cells contribute to disease processes. A popular method for distinguishing adventitial cell types relies on the use of genetic tools in the mouse to trace and manipulate these cells. Because lineage tracing relying on Cre-recombinase expressing mice is used more frequently in studies of vascular disease, it is important to outline the advantages and limitations of these genetic tools. The purpose of this article is to provide an overview of the various genetic tools available in the mouse for the study of resident adventitial fibroblasts.

Norepinephrine-evoked salt-sensitive hypertension requires impaired renal sodium chloride cotransporter activity in Sprague-Dawley rats.

Recent studies have implicated a role of norepinephrine (NE) in the activation of the sodium chloride cotransporter (NCC) to drive the development of salt-sensitive hypertension. However, the interaction between NE and increased salt intake on blood pressure remains to be fully elucidated. This study examined the impact of a continuous NE infusion on sodium homeostasis and blood pressure in conscious Sprague-Dawley rats challenged with a normal (NS; 0.6% NaCl) or high-salt (HS; 8% NaCl) diet for 14 days. Naïve and saline-infused Sprague-Dawley rats remained normotensive when placed on HS and exhibited dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide. NE infusion resulted in the development of hypertension, which was exacerbated by HS, demonstrating the development of the salt sensitivity of blood pressure [MAP (mmHg) NE+NS: 151 ± 3 vs. NE+HS: 172 ± 4; P < 0.05]. In these salt-sensitive animals, increased NE prevented dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide, suggesting impaired NCC activity contributes to the development of salt sensitivity [peak natriuresis to hydrochlorothiazide (µeq/min) Naïve+NS: 9.4 ± 0.2 vs. Naïve+HS: 7 ± 0.1; P < 0.05; NE+NS: 11.1 ± 1.1; NE+HS: 10.8 ± 0.4). NE infusion did not alter NCC expression in animals maintained on NS; however, dietary sodium-evoked suppression of NCC expression was prevented in animals challenged with NE. Chronic NCC antagonism abolished the salt-sensitive component of NE-mediated hypertension, while chronic ANG II type 1 receptor antagonism significantly attenuated NE-evoked hypertension without restoring NCC function. These data demonstrate that increased levels of NE prevent dietary sodium-evoked suppression of the NCC, via an ANG II-independent mechanism, to stimulate the development of salt-sensitive hypertension.

Revisiting Cardiac Cellular Composition.

RATIONALE: Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. OBJECTIVE: To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells, and fibroblasts in the mouse and human heart. METHODS AND RESULTS: Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute >60%, hematopoietic-derived cells 5% to 10%, and fibroblasts <20% of the nonmyocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of nonmyocytes. High-dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and 2 commonly assigned fibroblast markers, Sca-1 and CD90, under-represent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. CONCLUSIONS: This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of noncardiomyocytes and are likely to play a greater role in physiological function and response to injury than previously appreciated.

Gαi2-protein-mediated signal transduction: central nervous system molecular mechanism countering the development of sodium-dependent hypertension.

Excess dietary salt intake is an established cause of hypertension. At present, our understanding of the neuropathophysiology of salt-sensitive hypertension is limited by a lack of identification of the central nervous system mechanisms that modulate sympathetic outflow and blood pressure in response to dietary salt intake. We hypothesized that impairment of brain Gαi2-protein-gated signal transduction pathways would result in increased sympathetically mediated renal sodium retention, thus promoting the development of salt-sensitive hypertension. To test this hypothesis, naive or renal denervated Dahl salt-resistant and Dahl salt-sensitive (DSS) rats were assigned to receive a continuous intracerebroventricular control scrambled or a targeted Gαi2-oligodeoxynucleotide infusion, and naive Brown Norway and 8-congenic DSS rats were fed a 21-day normal or high-salt diet. High salt intake did not alter blood pressure, suppressed plasma norepinephrine, and evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi2-protein levels in naive Brown Norway, Dahl salt-resistant, and scrambled oligodeoxynucleotide-infused Dahl salt-resistant but not DSS rats. In Dahl salt-resistant rats, Gαi2 downregulation evoked rapid renal nerve-dependent hypertension, sodium retention, and sympathoexcitation. In DSS rats, Gαi2 downregulation exacerbated salt-sensitive hypertension via a renal nerve-dependent mechanism. Congenic-8 DSS rats exhibited sodium-evoked paraventricular nucleus-specific Gαi2-protein upregulation and attenuated hypertension, sodium retention, and global sympathoexcitation compared with DSS rats. These data demonstrate that paraventricular nucleus Gαi2-protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal nerve-dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Impairment of this mechanism contributes to the development of salt-sensitive hypertension.

Brain Gαi2-subunit protein-gated pathways are required to mediate the centrally evoked sympathoinhibitory mechanisms activated to maintain sodium homeostasis.

OBJECTIVE: We have previously demonstrated a role of GPCR-activated brain Gαi(2)-subunit protein-gated pathways in the natriuretic responses evoked by exogenous central α(2)-adrenoceptor activation and acute intravenous (i.v.) volume expansion in vivo. Our objective was to examine the role of brain Gαi(2) proteins in the integrated neural-humoral responses evoked by i.v. isovolumetric sodium loading, which does not alter mean arterial blood pressure or total blood volume, to maintain sodium homeostasis in conscious Sprague-Dawley rats. METHODS: Intact or chronic bilateral renal denervated (RDNX) rats were pretreated intracerebroventricularly (i.c.v.) with a scrambled or Gαi(2) oligodeoxynucleotide to selectively downregulate brain Gαi(2) proteins. On the day of study, an i.v. isovolumetric sodium load (1 mol/l NaCl) was administered. RESULTS: In naive and scrambled oligodeoxynucleotide groups, i.v. sodium loading evoked profound natriuresis, suppression of plasma renin activity (PRA) and global sympathoinhibition. Prior downregulation of brain Gαi(2) proteins significantly attenuated the natriuretic response [peak ΔUNaV (µeq/µl); scrambled 22 ± 2 vs. Gαi(2) 13 ± 2, P < 0.05] and abolished the sympathoinhibitory response [peak Δplasma norepinephrine (% control); SCR -72 ± 8 vs. Gαi(2) -7 ± 5, P < 0.05] without attenuating PRA suppression to sodium loading. In RDNX rats, Gαi(2) oligodeoxynucleotide pretreatment failed to attenuate the natriuretic response [peak ΔUNaV (µeq/µl); RDNX and scrambled 19 ± 3 vs. RDNX and Gαi(2) 20 ± 2] and only partially prevented the sympathoinhibitory response to i.v. sodium loading. CONCLUSION: These studies reveal a brain Gαi(2)-subunit protein-mediated (renin-angiotensin system-independent) sympathoinhibitory pathway that has a critical role in the central neural mechanisms activated to maintain fluid and electrolyte homeostasis.

Central nervous system Gαi2-subunit proteins maintain salt resistance via a renal nerve-dependent sympathoinhibitory pathway.

In salt-resistant phenotypes, chronic elevated dietary sodium intake evokes suppression of renal sodium-retaining mechanisms to maintain sodium homeostasis and normotension. We have recently shown that brain Gαi(2) protein pathways are required to suppress renal sympathetic nerve activity and facilitate maximal sodium excretion during acute intravenous volume expansion in Sprague-Dawley rats. Here, we studied the role of brain Gαi(2) proteins in the endogenous central neural mechanisms acting to maintain fluid and electrolyte homeostasis and normotension during a chronic elevation in dietary salt intake. Naive or bilaterally renal denervated adult male Sprague-Dawley rats were randomly assigned to receive an intracerebroventricular scrambled or Gαi(2) oligodeoxynucleotide infusion and then subjected to either a normal salt (0.4%) or high-salt (8.0%) diet for 21 days. In scrambled oligodeoxynucleotide-infused rats, salt loading, which did not alter blood pressure, evoked a site-specific increase in hypothalamic paraventricular nucleus Gαi(2) protein levels and suppression of circulating norepinephrine content and plasma renin activity. In salt-loaded rats continuously infused intracerebroventricularly with a Gαi(2) oligodeoxynucleotide, animals exhibited sodium and water retention, elevated plasma norepinephrine levels, and hypertension, despite suppression of plasma renin activity. Furthermore, in salt-loaded bilaterally renal denervated rats, Gαi(2) oligodeoxynucleotide infusion failed to evoke salt-sensitive hypertension. Therefore, in salt-resistant rats subjected to a chronic high-salt diet, brain Gαi(2) proteins are required to inhibit central sympathetic outflow to the kidneys and maintain sodium balance and normotension. In conclusion, these data demonstrate a central role of endogenous brain, likely paraventricular nucleus-specific, Gαi(2)-subunit protein-gated signal transduction pathways in maintaining a salt-resistant phenotype.