Ralph F. Johnson

Blood and urinary measures of hydration status during progressive acute dehydration

PURPOSE To determine whether: a) plasma osmolarity (Posm) is sensitive to small incremental changes in hydration status, b) urine specific gravity (Usg) can accurately identify a state of euhydration, c) Usg is a sensitive indicator of a change in hydration status, and d) Usg correlates with Posm. METHODS Euhydrated (Posm = 288 +/- 4 mOsm.L-1) subjects (N = 12) were dehydrated by 5% of their body weight via exercise in the heat (40 degrees C, 20% RH). Posm, urine osmolarity (Uosm), and Usg were measured at 1%, 3%, and 5% dehydration, and 30 and 60 min of recovery (rec). Subjects consumed water in recovery equal to their loss of body weight. RESULTS Posm increased incrementally with each successive increase in percent body weight loss (%BWL). Usg was not significantly different from baseline until 3% BML. Uosm was not significantly different from baseline until 5% BWL. Usg correlated moderately (r = 0.46, P > 0.10) with Posm but reasonably well (r = 0.68, P < 0.02) with Uosm. CONCLUSIONS Posm accurately identifies a state of euhydration and is sensitive to changes in hydration status during acute dehydration and rehydration. Usg and Uosm are also sensitive to changes in hydration status but lag behind during periods of rapid body fluid turnover and therefore correlate only moderately with Posm during acute dehydration.

Novel Effect of Mineralocorticoid Receptor Antagonism to Reduce Proinflammatory Cytokines and Hypothalamic Activation in Rats With Ischemia-Induced Heart Failure

Blocking brain mineralocorticoid receptors (MRs) reduces the high circulating levels of tumor necrosis factor (TNF)-&agr; in heart failure (HF) rats. TNF-&agr; and other proinflammatory cytokines activate neurons in the paraventricular nucleus (PVN) of hypothalamus, including corticotropin-releasing hormone (CRH) neurons, by inducing cyclooxygenase (COX)-2 activity and synthesis of prostaglandin E2 by perivascular cells of the cerebral vasculature. We tested the hypothesis that systemic treatment with a MR antagonist would reduce hypothalamic COX-2 expression and PVN neuronal activation in HF rats. Rats underwent coronary ligation to induce HF, confirmed by echocardiography, or sham surgery, followed by 6 weeks treatment with eplerenone (30 mg/kg per day, orally) or vehicle (drinking water). Eplerenone-treated HF rats had lower plasma TNF-&agr;, interleukin (IL)-1&bgr; and IL-6, less COX-2 staining of small blood vessels penetrating PVN, fewer PVN neurons expressing Fra-like activity (indicating chronic neuronal activation), and fewer PVN neurons staining for TNF-&agr;, IL-1&bgr;, and CRH than vehicle-treated HF rats. COX-2 and CRH protein expression in hypothalamus were 1.7- and 1.9-fold higher, respectively, in HF+vehicle versus sham+vehicle rats; these increases were attenuated (26% and 25%, respectively) in HF+eplerenone rats. Eplerenone-treated HF rats had less prostaglandin E2 in cerebrospinal fluid, lower plasma norepinephrine levels, lower left ventricular end-diastolic pressure, and lower right ventricle/body weight and lung/body weight ratios, but no improvement in left ventricular function. Treatment of HF rats with anticytokine agents, etanercept or pentoxifylline, produced very similar results. This study reveals a previously unrecognized effect of MR antagonism to minimize cytokine-induced central neural excitation in rats with HF.

The neural substrates of enhanced salt appetite after repeated sodium depletions.

Sodium appetite is associated with a form of behavioral plasticity in which animals experimentally depleted of sodium progressively increase their intake of hypertonic NaCl over several successive (on 2 to 4 occasions) depletion. The present experiment explored the nature of this plasticity by quantifying Fos immunoreactivity (Fos-ir) in structures implicated in the mediation of sodium appetite and in the signaling of reward. Rats were depleted of sodium with the diuretic furosemide three times (3F), one time (2V1F) or sham depleted (i.e., vehicle treated; 3V). Rats were given sodium appetite tests for the first two treatments. The sodium appetite test was omitted after the third treatment. Fos-ir activity was quantified in the paraventricular nucleus (PVN), subfornical organ (SFO), supraoptic nucleus (SON), nucleus accumbens (NAc) shell and core, basolateral (BLA) and central amygdala (CeA), and medial prefrontal cortex (mPFC). Animals receiving repeated sodium depletions increased sodium ingestion across initial depletions. Fos-ir activity was markedly enhanced in the SFO, BLA, and shell of the NAc of 3F rats relative to 2V1F and 3V animals. These results indicate that repeated experience with sodium depletion and ingestion affects both behavioral and neural responses to sodium. Experience with sodium depletion enhances its ingestion and may have a direct impact on central structures implicated in sodium appetite and reward signaling.

Sensitization of Slow Pressor Angiotensin II (Ang II)–Initiated Hypertension: Induction of Sensitization by Prior Ang II Treatment

Sensitization involving the central nervous system has been studied in many conditions but has received little attention in investigation of the pathogenesis of hypertension. Our experiments were initiated to determine whether angiotensin II (Ang II)–induced hypertension can be sensitized by prior Ang II treatment and the role of the brain renin-angiotensin-aldosterone system (RAAS) in this process. To demonstrate Ang II–induced sensitization, we used an experimental design of induction-delay-expression. Male rats were implanted for telemetered blood pressure (BP) recording. During induction (I), low doses of subcutaneous or intracerebroventricular Ang II were delivered for 1 week, and then the rats were rested for 1 week (delay [D]) to ensure that any exogenous Ang II was metabolized. After this, a second higher dose of Ang II was given subcutaneously for 2 weeks (expression [E]). During I and D, the low doses of Ang II had no sustained effects on BP. However, during E, the Ang II–induced BP increase was greater in the groups that had received low doses of Ang II during I in comparison to the group receiving saline during I. Central angiotensin type 1 receptor antagonist delivery blocked this sensitization. Brain tissue collected at the end of D and E showed increased mRNA expression of several RAAS components in key forebrain regions of sensitized rats. Fos-related antigen–like immunoreactivity was also increased at the end of E in the sensitized forebrain. These results indicate that subpressor doses of Ang II act on the brain to sensitize the hypertensive response to subsequent Ang II and that sensitization is associated with altered expression of RAAS components in forebrain cardiovascular control structures.

The effects of ibotenate lesions of the median preoptic nucleus on experimentally-induced and circadian drinking behavior in rats

Male Sprague-Dawley rats were injected with either ibotenic acid or vehicle in the region of the median preoptic nucleus (MnPO) during methoxyflurane anesthesia. Later, the rats were tested for drinking responses elicited by angiotensin II (1 and 2 mg/kg s.c.) and hypertonic saline (3 and 6% w/v s.c.). Tests were conducted at 3 different phases of a 12:12 light-dark (LD) cycle (in the middle of the light phase and in the early and midportion of the dark phase). The rats with ibotenate lesions of the MnPO drank significantly less than vehicle-injected and lesion control groups regardless of when the tests were conducted. Subsequent monitoring of the diurnal rhythm of drinking, employing electrical lickometers, detected no difference between the rhythms of 4 rats with ibotenate lesions of the MnPO who failed to drink to homeostatic challenges and 4 vehicle-injected control rats. The results indicate that ibotenic acid lesions of the MnPO block drinking behavior stimulated by angiotensin II and hypertonic saline without disrupting the entrainment or pattern of ad libitum drinking.

PVN adenovirus-siRNA injections silencing either NOX2 or NOX4 attenuate aldosterone/NaCl-induced hypertension in mice

Mineralocorticoid excess increases superoxide production by activating NADPH oxidase (NOX), and intracerebroventricular infusions of NADPH oxidase inhibitors attenuate aldosterone (Aldo)/salt-induced hypertension. It has been hypothesized that increased reactive oxygen species (ROS) in the brain may be a key mechanism in the development of hypertension. The present study investigated the brain regional specificity of NADPH oxidase and the role of NOX2 and NOX4 NADPH oxidase subunits in the hypothalamic paraventricular nucleus (PVN) in Aldo/salt-induced hypertension. PVN injections of adenoviral vectors expressing small interfering (si)RNA targeting NOX2 (AdsiRNA-NOX2) or NOX4 (AdsiRNA-NOX4) mRNAs were used to knock down NOX2 and NOX4 proteins. Three days later, delivery of Aldo (0.2 mg·kg(-1)·day(-1) sc) via osmotic pump commenced and 1% NaCl was provided in place of water. PVN injections of either AdsiRNA-NOX2 or AdsiRNA-NOX4 significantly attenuated the development of Aldo/NaCl-induced hypertension. In an additional study, Aldo/salt-induced hypertension was also significantly attenuated in NOX2 (genomic) knockout mice compared with wild-type controls. When animals from both functional studies underwent ganglionic blockade, there was a reduced fall in blood pressure in the NOX2 and NOX4 knockdown/knockout mice. Western blot analyses of the PVN of siRNA-NOX2- or siRNA-NOX4-injected mice confirmed a marked reduction in the expression of NOX2 or NOX4 protein. In cultured PVN neurons, silencing either NOX2 or NOX4 protein production by culturing PVN cells with siRNA-NOX2 or siRNA-NOX4 attenuated Aldo-induced ROS. These data indicate that both NOX2 and NOX4 in the PVN contribute to elevated sympathetic activity and the hypertensivogenic actions induced by mineralocorticoid excess.

Estrogen Receptor-β in the Paraventricular Nucleus and Rostroventrolateral Medulla Plays an Essential Protective Role in Aldosterone/Salt-Induced Hypertension in Female Rats

The identification of the specific estrogen receptor (ER) subtypes that are involved in estrogen protection from hypertension and their specific locations in the central nervous system is critical to our understanding and design of effective estrogen replacement therapies in women. Using selective ER agonists and recombinant adeno-associated virus (AAV) carrying small interference (si) RNA to silence either ER&agr; (AAV-siRNA-ER&agr;) or ER&bgr; (AAV-siRNA-ER&bgr;), the present study investigated regional specificity of different ER subtypes in the protective actions of estrogen in aldosterone (Aldo)-induced hypertension. Intracerebroventricular infusions of either diarylpropionitrile, a selective ER&bgr; agonist, or propyl-pyrazole-triol, a selective ER&agr; agonist, attenuated Aldo/NaCl-induced hypertension in ovariectomized rats. In contrast, intracerebroventricular injections of siRNA-ER&agr; or siRNA-ER&bgr; augmented Aldo-induced hypertension in intact females. Site-specific paraventricular nucleus (PVN) or rostroventrolateral medulla (RVLM) injections of siRNA-ER&bgr; augmented Aldo-induced hypertension. However, rats with PVN or RVLM injections of siRNA-ER&agr; did not significantly increase blood pressure induced by Aldo. Real-time polymerase chain reaction analyses of the PVN and RVLM of siRNA-injected rat confirmed a marked reduction in the expression of ER&agr; and ER&bgr;. In cultured PVN neurons, silencing either ER&agr; or ER&bgr; by culturing PVN neurons with siRNA-ER&agr; or siRNA-ER&bgr; enhanced Aldo-induced reactive oxygen species production. Ganglionic blockade after Aldo infusion showed an increase in sympathetic activity in ER&bgr; knockdown rats. These results indicate that both PVN and RVLM ER&bgr;, but not ER&agr; in these nuclei, contribute to the protective effects of estrogen against Aldo-induced hypertension. The brain regions responsible for the protective effects of estrogen interaction with ER&agr; in Aldo-induced hypertension still need to be determined.

Non-NMDA receptors in the lateral parabrachial nucleus modulate sodium appetite.

Glutamatergic mechanisms have been implicated in the control of fluid ingestion. In the present study, we investigated whether non-N-methyl-d-aspartate (NMDA) glutamatergic receptors in the lateral parabrachial nucleus (LPBN) are involved in the control of water and sodium intake. Male Sprague-Dawley rats had cannulas implanted bilaterally into the LPBN. They were acutely depleted of water and sodium by injections of the diuretic furosemide (Furo; 10 mg/kg, bw) and given a low dose of the angiotensin-converting enzyme inhibitor, captopril (Cap; 5 mg/kg, bw). Bilateral LPBN injections of the non-NMDA receptor antagonist DNQX (2 and 5 nmol/0.2 microl) increased the ingestion of 0.3 M NaCl and water of Furo/Cap treated rats. The increased ingestion produced by DNQX was abolished by pretreating the LPBN with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), a non-NMDA receptor agonist. AMPA injected alone into the LPBN reduced water and 0.3 M NaCl intake. Injections of DNQX (5 nmol/0.2 microl) into the LPBN also produced ingestion of 0.3 M NaCl after sc injections of the beta-adrenoceptor agonist, isoproterenol, a hypotensive drug that typically produces only water intake. Food intake, arterial blood pressure and heart rate were not altered by DNQX LPBN injections. We conclude that agonists acting on non-NMDA receptors in the LPBN exert an inhibitory influence on sodium intake during acute fluid depletion with hypotension and after isoproterenol treatment. A possible interaction of serotonin with glutamate within the LPBN is discussed.

Light/dark cycle modulates food to water intake ratios in rats.

Ad lib water intake of rats displays two prominent features: Water intake is temporally and quantitatively associated with food intake, and water intake is nocturnal. Physiological mechanisms linking water and food intake suggest that the rhythmicity in water intake may be at least partially driven by a rhythmicity in food intake. The present study examined whether the daily rhythm of rats modulates the association of food and water intake. Rats were housed under light/dark cycles and continuously monitored for temporal and quantitative patterns of ad lib food and water intake. The results indicate that the water/food ratio is much lower for meals consumed in the light phase than in the dark phase. The above results suggest a rhythmic modulation of the mechanisms linking food and water intake. We suggest that much of the complex rhythm of drinking stems from several interacting factors. 1) Food intake is nocturnal. 2) Food intake induces water intake by physiological mechanisms. 3) The amount of water intake induced by food intake is influenced by rhythmic factors.

Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis.