AJP: Regulatory, Integrative and Comparative Physiology current issue

VEGF kinase inhibitors: how do they cause hypertension?

Bhargava, P. — 2009-07-02

Neoangiogenesis is a critical phenomenon enabling the growth and metastasis of tumors, and inhibitors of neoangiogenesis have been recently added to the armamentarium of anticancer therapies available for clinical use. Dysregulated signaling through the vascular endothelial growth factor (VEGF) pathway has been implicated as a key mediator of neoangiogenesis in tumors. Agents that block signaling through the VEGF pathway demonstrated tumor shrinkage in preclinical models and were therefore developed as anticancer therapies for use in humans. VEGF kinase inhibitors are being used in the treatment of a wide variety of cancers, and recent studies have shown that patients will likely require long-term treatment with these agents. Hypertension has emerged as a frequent side effect associated with agents that block signaling through the VEGF pathway. A thorough understanding of the mechanisms underlying hypertension is crucial to developing appropriate therapeutic strategies for treating hypertension associated with VEGF kinase inhibitors. Several recent studies have advanced our understanding of the pathophysiology of hypertension associated with VEGF kinase inhibitors and will be the subject of this review.

Balance point characterization of interstitial fluid volume regulation

Dongaonkar, R. M., Laine, G. A., Stewart, R. H., Quick, C. M. — 2009-07-02

The individual processes involved in interstitial fluid volume and protein regulation (microvascular filtration, lymphatic return, and interstitial storage) are relatively simple, yet their interaction is exceedingly complex. There is a notable lack of a first-order, algebraic formula that relates interstitial fluid pressure and protein to critical parameters commonly used to characterize the movement of interstitial fluid and protein. Therefore, the purpose of the present study is to develop a simple, transparent, and general algebraic approach that predicts interstitial fluid pressure (P_i) and protein concentrations (C_i) that takes into consideration all three processes. Eight standard equations characterizing fluid and protein flux were solved simultaneously to yield algebraic equations for P_i and C_i as functions of parameters characterizing microvascular, interstitial, and lymphatic function. Equilibrium values of P_i and C_i arise as balance points from the graphical intersection of transmicrovascular and lymph flows (analogous to Guyton's classical cardiac output-venous return curves). This approach goes beyond describing interstitial fluid balance in terms of conservation of mass by introducing the concept of inflow and outflow resistances. Algebraic solutions demonstrate that P_i and C_i result from a ratio of the microvascular filtration coefficient (1/inflow resistance) and effective lymphatic resistance (outflow resistance), and P_i is unaffected by interstitial compliance. These simple algebraic solutions predict P_i and C_i that are consistent with reported measurements. The present work therefore presents a simple, transparent, and general balance point characterization of interstitial fluid balance resulting from the interaction of microvascular, interstitial, and lymphatic function.

Microbial experimental evolution

Bennett, A. F., Hughes, B. S. — 2009-07-02

Microbes have been widely used in experimental evolutionary studies because they possess a variety of valuable traits that facilitate large-scale experimentation. Many replicated populations can be cultured in the laboratory simultaneously along with appropriate controls. Short generation times and large population sizes make microbes ideal experimental subjects, ensuring that many spontaneous mutations occur every generation and that adaptive variants can spread rapidly through a population. Another highly useful experimental feature is the ability to preserve and store ancestral and evolutionarily derived clones. These can be revived in parallel to allow the direct measurement of the competitive fitness of a descendant compared with its ancestor. The extent of adaptation can thereby be measured quantitatively and compared statistically by direct competition among derived groups and with the ancestor. Thus, fitness and adaptation need not be matters of qualitative speculation, but are quantitatively measurable variables in these systems. Replication allows the quantification of heterogeneity in responses to imposed selection and thereby statistical distinction between changes that are systematic responses to the selective regimen and those that are specific to individual populations.

Temporary fatigue and altered extracellular matrix in skeletal muscle during progression of heart failure in rats

2009-07-02

Patients with congestive heart failure (CHF) experience increased skeletal muscle fatigue. The mechanism underlying this phenomenon is unknown, but a deranged extracellular matrix (ECM) might be a contributing factor. Hence, we examined ECM components and regulators in a rat postinfarction model of CHF. At various time points during a 3.5 mo-period after induction of CHF in rats by left coronary artery ligation, blood, interstitial fluid (IF), and muscles were sampled. Isoflurane anesthesia was employed during all surgical procedures. IF was extracted by wicks inserted intermuscularly in a hind limb. We measured cytokines in plasma and IF, whereas matrix metalloproteinase (MMP) activity and collagen content, as well as the level of glycosaminoglycans and hyaluronan were determined in hind limb muscle. In vivo fatigue protocols of the soleus muscle were performed at 42 and 112 days after induction of heart failure. We found that the MMP activity and collagen content in the skeletal muscles increased significantly at 42 days after induction of CHF, and these changes were time related to increased skeletal muscle fatigability. These parameters returned to sham levels at 112 days. VEGF in IF was significantly lower in CHF compared with sham-operated rats at 3 and 10 days, but no difference was observed at 112 days. We conclude that temporary alterations in the ECM, possibly triggered by VEGF, are related to a transient development of skeletal muscle fatigue in CHF.

Dorsomedial medullary 5-HT2 receptors mediate immediate onset of initial hyperventilation, airway dilation, and ventilatory decline during hypoxia in mice

Kanamaru, M., Homma, I. — 2009-07-02

The dorsomedial medulla oblongata (DMM) includes the solitary tract nucleus and the hypoglossal nucleus, to which 5-HT neurons project. Effects of 5-HT in the DMM on ventilatory augmentation and airway dilation are mediated via 5-HT2 receptors, which interact with the CO₂ drive. The interaction may elicit cycles between hyperventilation with airway dilation and hypoventilation with airway narrowing. In the present study, effects of 5-HT2 receptors in the DMM on hypoxic ventilatory and airway responses were investigated, while 5-HT release in the DMM was monitored. Adult male mice were anesthetized, and then a microdialysis probe was inserted into the DMM. The mice were placed in a double-chamber plethysmograph. After recovery from anesthesia, the mice were exposed to hypoxic gas (7% O₂ in N₂) for 5 min with or without a 5-HT2 receptor antagonist (LY-53857) perfused in the DMM. 5-HT release in the DMM was increased by hypoxia regardless of the presence of LY-53857. Immediate onset and the peak of initial hypoxic hyperventilatory responses were delayed. Subsequent ventilatory decline and airway dilation during initial hypoxic hyperventilation were suppressed with LY-53857. These results suggest that 5-HT release increased by hypoxia acts on 5-HT2 receptors in the DMM, which contributes to the immediate onset of initial hypoxic hyperventilation, airway dilation, and subsequent ventilatory decline. Hypoxic ventilatory and airway responses mediated via 5-HT2 receptors in the DMM may play roles in immediate rescue and defensive adaptation for hypoxia and may be included in periodic breathing and the pathogenesis of obstructive sleep apnea.

Cortisol and corticosterone in immune organs and brain of European starlings: developmental changes, effects of restraint stress, comparison with zebra finches

Schmidt, K. L., Chin, E. H., Shah, A. H., Soma, K. K. — 2009-07-02

Glucocorticoids (GCs) are produced in the adrenal glands and also in extra-adrenal sites, including immune organs and brain. Here, we examined regulation of systemic GC levels in plasma and local GC levels in immune organs and brain during development. We conducted two studies and examined a total of 462 samples from 70 subjects. In study 1, we determined corticosterone and cortisol levels in the plasma, immune organs, and brain of wild European starlings on posthatch day 0 (P0) and P10 (at baseline and after 45 min of restraint). Baseline corticosterone and cortisol levels were low in the immune organs and brain at P0 and P10, providing little evidence for local GC synthesis in starlings. At P0, restraint had no significant effects on corticosterone or cortisol levels in the plasma or tissues; however, there was a trend for restraint to increase both corticosterone and cortisol in the immune organs. At P10, restraint increased corticosterone levels in the plasma and all tissues, but restraint increased cortisol levels in the plasma, thymus, and diencephalon only. In study 2, we directly compared GC levels in European starlings and zebra finches at P4. In zebra finches but not starlings, cortisol levels were higher in the immune organs than in plasma. This difference in immune GC levels might be due to evolutionary lineage, life history strategy, or experiential factors, such as parasite exposure. This is the first study to measure immune GC levels in wild animals and one of the first studies to measure local GC levels after restraint stress.

Enhancement of Ih, but not inhibition of IM, is a key mechanism underlying the PACAP-induced increase in excitability of guinea pig intrinsic cardiac neurons

Tompkins, J. D., Lawrence, Y. T., Parsons, R. L. — 2009-07-02

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated by PACAP-selective PAC₁ receptors. In dissociated guinea pig cardiac neurons, PACAP causes a positive shift of the voltage dependence of activation of the hyperpolarization-activated nonselective cation current (I_h). This observation suggested that an enhancement of I_h contributed to the increase in excitability in neurons within whole-mount cardiac ganglia preparations. To evaluate the role of I_h in the PACAP-induced increase in excitability, we compared the increase in action potentials generated by 10 nM PACAP in control neurons and in neurons treated with ZD7288 (10 or 100 µM) or CsCl (2 or 2.5 mM), drugs known to inhibit I_h. In control cells exposed to PACAP, 1-s depolarizing current pulses elicited multiple action potential firing in 79% of the neurons. In ZD7288- or CsCl-containing solutions, the 10 nM PACAP-induced increase in excitability was markedly suppressed, with 7% and 21% of the neurons generating multiple action potentials, respectively. Prior results indicated that PACAP initiates depolarization by activating an inward current, which is separate from its enhancement of I_h. Here, we show that a PACAP-induced depolarization was comparable in control neurons and neurons bathed in a CsCl-containing solution, an observation indicating that CsCl did not interfere with activation of the PAC₁ receptor by PACAP. Additional experiments indicated that pretreatment with the putative M current (I_M) inhibitor 1 mM BaCl₂, but not 10 µM XE991, initiated multiple firing in a majority of neurons, with resting potentials maintained at approximately –60 mV. Furthermore, in Ba²⁺-treated cells, 10 nM PACAP increased the number of action potentials generated. Our results indicate that PACAP enhancement of I_h, rather than inhibition of I_M and other 1 mM Ba²⁺-sensitive K⁺ currents, is a key ionic mechanism contributing to the peptide-induced increase in excitability for neurons within whole-mount cardiac ganglia preparations.

Changes in cerebral blood flow, cerebral metabolites, and breathing movements in the sheep fetus following asphyxia produced by occlusion of the umbilical cord

Yan, E. B., Baburamani, A. A., Walker, A. M., Walker, D. W. — 2009-07-02

Severe global fetal asphyxia, if caused by a brief occlusion of the umbilical cord, results in prolonged cerebral hypoperfusion in fetal sheep. In this study, we sought evidence to support the hypothesis that cerebral hypoperfusion is a consequence of suppressed cerebral metabolism. In the 24 h following complete occlusion of the umbilical cord for 10 min, sagittal sinus blood flow velocity was significantly decreased for up to 12 h. Capillary blood flow, measured using microspheres, decreased at 1 and 5 h after cord occlusion in many brain regions, including cortical gray and white matter. Microdialysis probes implanted in the cerebral cortex revealed an increase in extracellular glucose concentrations in gray matter for 7–8 h postasphyxia, while lactate increased only briefly, suggesting decreased cerebral glucose utilization over this time. Although these data, as well as the concurrent suppression of breathing movements and electrocortical activity, support the concept of hypometabolic hypoperfusion, the significant increase of pyruvate and glycerol concentrations in dialysate fluid obtained from the cerebral cortex at 3–8 h after cord occlusion suggests an eventual loss of membrane integrity. The prolonged increase of breathing movements for many hours suggests loss of the pontine/thalamic control that produces the distinct pattern of fetal breathing movements.

Short-term blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharide-stimulated inflammatory responses

2009-07-02

Exercise-induced oxidative stress is instrumental in achieving the health benefits from regular exercise. Therefore, inappropriate use of fruit-derived products (commonly applied as prophalytic antioxidants) may counteract the positive effects of exercise. Using human exercise and cellular models we found that 1) blackcurrant supplementation suppressed exercise-induced oxidative stress, e.g., plasma carbonyls (0.9 ± 0.1 vs. 0.6 ± 0.1 nmol/mg protein, placebo vs. blackcurrant), and 2) preincubation of THP-1 cells with an anthocyanin-rich blackcurrant extract inhibited LPS-stimulated cytokine secretion [TNF- (16,453 ± 322 vs. 10,941 ± 82 pg/ml, control vs. extract, P < 0.05) and IL-6 (476 ± 14 vs. 326 ± 32 pg/ml, control vs. extract, P < 0.05)] and NF-B activation. In addition to its antioxidant and anti-inflammatory properties, we found that postexercise plasma collected after blackcurrant supplementation enhanced the differential temporal LPS-stimulated inflammatory response in THP-1 cells, resulting in an early suppression of TNF- (1,741 ± 32 vs. 1,312 ± 42 pg/ml, placebo vs. blackcurrant, P < 0.05) and IL-6 (44 ± 5 vs. 36 ± 3 pg/ml, placebo vs. blackcurrant, P < 0.05) secretion after 24 h. Furthermore, by using an oxidative stress cell model, we found that preincubation of THP-1 cells with hydrogen peroxide (H₂O₂) prior to extract exposure caused a greater suppression of LPS-stimulated cytokine secretion after 24 h, which was not evident when cells were simultaneously incubated with H₂O₂ and the extract. In summary, our findings support the concept that consumption of blackcurrant anthocyanins alleviate oxidative stress, and may, if given at the appropriate amount and time, complement the ability of exercise to enhance immune responsiveness to potential pathogens.

SOD1 deficiency causes salt sensitivity and aggravates hypertension in hydronephrosis

2009-07-02

Hydronephrosis causes renal dysfunction and salt-sensitive hypertension, which is associated with nitric oxide deficiency and abnormal tubuloglomerular feedback (TGF) response. We investigated the role of oxidative stress for salt sensitivity and for hypertension in hydronephrosis. Hydronephrosis was induced in superoxide dismutase 1-transgenic (SOD1-tg), SOD1-deficient (SOD1-ko), and wild-type mice and in rats. In mice, telemetric measurements were performed during normal (0.7% NaCl) and high-sodium (4% NaCl) diets and with chronic tempol supplementation. The 8-iso-prostaglandin-F₂ (F2-IsoPs) and protein excretion profiles and renal histology were investigated. The acute effects of tempol on blood pressure and TGF were studied in rats. In hydronephrosis, wild-type mice developed salt-sensitive hypertension (114 ± 1 to 120 ± 2 mmHg), which was augmented in SOD1-ko (125 ± 3 to 135 ± 4 mmHg) but abolished in SOD1-tg (109 ± 3 to 108 ± 3 mmHg). SOD1-ko controls displayed salt-sensitive blood pressure (108 ± 1 to 115 ± 2 mmHg), which was not found in wild types or SOD1-tg. Chronic tempol treatment reduced blood pressure in SOD1-ko controls (–7 mmHg) and in hydronephrotic wild-type (–8 mmHg) and SOD1-ko mice (–16 mmHg), but had no effect on blood pressure in wild-type or SOD1-tg controls. SOD1-ko controls and hydronephrotic wild-type and SOD1-ko mice exhibited increased fluid excretion associated with increased F2-IsoPs and protein excretion. The renal histopathological changes found in hydronephrotic wild-type were augmented in SOD1-ko and diminished in SOD-tg mice. Tempol attenuated blood pressure and normalized TGF response in hydronephrosis [P_SF: 15.2 ± 1.2 to 9.1 ± 0.6 mmHg, turning point: 14.3 ± 0.8 to 19.7 ± 1.4 nl/min]. Oxidative stress due to SOD1 deficiency causes salt sensitivity and plays a pivotal role for the development of hypertension in hydronephrosis. Increased superoxide formation may enhance TGF response and thereby contribute to hypertension.

Prenatal programming of rat thick ascending limb chloride transport by low-protein diet and dexamethasone

Dagan, A., Habib, S., Gattineni, J., Dwarakanath, V., Baum, M. — 2009-07-02

Prenatal administration of dexamethasone and a low-protein diet has been shown to result in hypertension in the offspring when they are adults. The cause for the hypertension is unknown. The purpose of this study was to examine whether there was prenatal programming of thick ascending limb transport. Rats were administered either dexamethasone for 4 days (0.2 mg/kg body wt) by intraperitoneal injection daily between the 15th and 18th day of gestation, or they were fed a low-protein diet (6% protein) or an isocaloric normal protein diet (20% protein) from day 12 gestation until birth. The offspring were studied as adults. Prenatal dexamethasone and dietary protein deprivation resulted in an increase in blood pressure. Offspring of mothers fed a low-protein diet had an increase in medullary but not cortical bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) protein abundance (P < 0.01). There was not a statistically significant increase in medullary NKCC2 by prenatal dexamethasone (P = 0.07). Both prenatal administration of dexamethasone and a low-protein diet resulted in an increase in medullary thick ascending limb chloride transport compared with control (298 ± 33 pmoles·mm^–1·min^–1, 280 ± 26 pmoles·mm^–1·min^–1, and 191 ± 21 pmoles·mm^–1·min^–1, respectively P < 0.05). There was a higher lumen-positive transepithelial potential difference in the prenatal dexamethasone and low-protein group compared with control as well. Administration of furosemide for 24 h resulted in a decrease in blood pressure in the low-protein group but not the control group. This study demonstrates that insults administered to the fetus can program altered sodium transport. Increased tubular sodium transport is a likely cause for the hypertension by prenatal programming.

Diet-derived nutrients mediate the inhibition of hypothalamic NPY neurons in the arcuate nucleus of mice during refeeding

Becskei, C., Lutz, T. A., Riediger, T. — 2009-07-02

Fasting activates orexigenic neuropeptide Y neurons in the hypothalamic arcuate nucleus (ARC) of mice, which is reversed by 2 h refeeding with standard chow. Here, we investigated the contribution of diet-derived macronutrients and anorectic hormones to the reversal of the fasting-induced ARC activation during 2 h refeeding. Refeeding of 12-h-fasted mice with a cellulose-based, noncaloric mash induced only a small reduction in c-Fos expression. Refeeding with diets, containing carbohydrates, protein, or fat alone reversed it similar to chow; however, this effect depended on the amount of intake. The fasting-induced ARC activation was unchanged by subcutaneously injected amylin, CCK (both 20 µg/kg), insulin (0.2 U/kg and 0.05 U/kg) or leptin (2.6 mg/kg). Insulin and leptin had no effect on c-Fos expression in neuropeptide Y or proopiomelanocortin-containing ARC neurons. Interestingly, CCK but not amylin reduced the ghrelin-induced c-Fos expression in the ARC in ad libitum-fed mice, suggesting that CCK may inhibit orexigenic ARC neurons when acting together with other feeding-related signals. We conclude that all three macronutrients and also non-nutritive, ingestion-dependent signals contribute to an inhibition of orexigenic ARC neurons after refeeding. Similar to the previously demonstrated inhibitory in vivo action of peptide YY, CCK may be a postprandial mediator of ARC inhibition.

Chronic immunoneutralization of brain angiotensin-(1-12) lowers blood pressure in transgenic (mRen2)27 hypertensive rats

2009-07-02

Angiotensin-(1-12) [ANG-(1-12)] is a newly identified peptide detected in a variety of rat tissues, including the brain. To determine whether brain ANG-(1-12) participates in blood pressure regulation, we treated male adult (mRen2)27 hypertensive rats (24–28 wk of age) with Anti-ANG-(1-12) IgG or Preimmune IgG via an intracerebroventricular cannula for 14 days. Immunoneutralization of brain ANG-(1-12) lowered systolic blood pressure (–43 ± 8 mmHg on day 3 and –26 ± 7 mmHg on day 10 from baseline, P < 0.05). Water intake was lower on intracereroventricular day 6 in the Anti-ANG-(1-12) IgG group, accompanied by higher plasma osmolality on day 13, but there were no differences in urine volume, food intake, or body weight during the 2-wk treatment. In Preimmune IgG-treated animals, there were no significant changes in these variables over the 2-wk period. The antihypertensive effects produced by endogenous neutralization of brain ANG-(1-12) suggest that ANG-(1-12) is functionally active in brain pathways regulating blood pressure.

Arterial-cardiac baroreflex function: insights from repeated squat-stand maneuvers

2009-07-02

To assess baroreflex function under closed-loop conditions, a new approach was used to generate large and physiological perturbations in arterial pressure. Blood pressure (BP) and R-R interval were recorded continuously in 20 healthy young (33 ± 8 yr) and eight elderly subjects (66 ± 6 yr). Repeated squat-stand maneuvers at the frequencies of 0.05 and 0.1 Hz were performed to produce periodic oscillations in BP to provoke the baroreflex. To assess the effects of the muscle reflex and/or central command on the baroreflex, passive squat-stand maneuvers were conducted using a pulley system to assist changes in body position. Transfer function between changes in BP and R-R interval was estimated to assess the arterial-cardiac baroreflex. Relative to resting conditions, large and coherent oscillations in BP and R-R interval were produced during both active and passive squat-stand maneuvers. However, changes in BP were smaller during passive than during active maneuvers. Changes in R-R interval were reduced commensurately. Therefore, transfer function gain did not change between the two maneuvers. Compared with the young, transfer function gain was reduced and the phase became more negative in the elderly, demonstrating the well-known effects of aging on reducing baroreflex sensitivity. Collectively, these findings suggest that the changes in R-R interval elicited by BP perturbations during squat-stand maneuvers are mediated primarily by a baroreflex mechanism. Furthermore, baroreflex function can be assessed using the transfer function method during large perturbations in arterial pressure.

Breath-to-breath hypercapnic response in neonatal rats: temperature dependency of the chemoreflexes and potential implications for breathing stability

Cummings, K. J., Frappell, P. B. — 2009-07-02

The breathing of newborns is destabilized by warm temperatures. We hypothesized that in unanesthetized, intact newborn rats, body temperature (T_B) influences the peripheral chemoreflex response (PCR response) to hypercapnia. To test this, we delivered square-wave challenges of 8% CO₂ in air to postnatal day 4–5 (P4–P5) rats held at a T_B of 30°C (Cold group, n = 11), 33°C (Cool group, n = 10), and 35°C thermoneutral zone group [thermoneutral zone (TNZ) group, n = 11], while measuring ventilation (Ve) directly with a pneumotach and mask. Cool animals were challenged with 8% CO₂ balanced in either air or hyperoxia (n = 10) to identify the PCR response. Breath-to-breath analysis was performed on 30 room air breaths and every breath of the 1-min CO₂ challenge. As expected, warmer T_B was associated with an unstable breathing pattern in room air: TNZ animals had a coefficient of variation in Ve (Ve CV%) that was double that of animals held at cooler T_B (P < 0.001). Hyperoxia markedly suppressed the hypercapnic ventilatory response over the first 10 breaths (or ~4 s), suggesting that this domain is dominated by the PCR response. The PCR response (P = 0.03) and total response (P = 0.04) were significantly greater in TNZ animals compared with hypothermic animals. The total response had a significant, negative relationship with Vco₂ (R² = 0.53; P < 0.001). Breathing stability was positively related to the total response (R² = 0.36; P < 0.001) and to a lesser extent, the PCR response (R² = 0.19; P = 0.01) and was negatively related to Vco₂ (R² = 0.34; P < 0.001). ANCOVA confirmed a significant effect of T_B alone on breathing stability (P < 0.01), with no independent effects of Vco₂ (P = 0.41), the PCR response (P = 0.82), or the total Ve response (P = 0.08). Our data suggest that in early postnatal life, the chemoreflex responses to CO₂ are highly influenced by T_B, and while related to breathing stability, are not predictors of stability after accounting for the independent effect of T_B.

Social cues from conspecifics alter electrical activity of gonadotropin-releasing hormone neurons in the terminal nerve via visual signals

Ramakrishnan, S., Wayne, N. L. — 2009-07-02

There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons in the brains of vertebrates. The population located in the hypothalamus/preoptic area is the best studied and is known to ultimately control reproduction. Teleost fish have an additional population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulbs, the physiological function of which is still unclear. Anatomical and physiological studies provide evidence that TN-GnRH neurons have extensive projections in the brain and modulate neuronal activity. Although there is anatomical evidence that the TN receives olfactory and optic sensory inputs, it is not known if sensory information is transmitted to TN-GnRH neurons to modulate their activity. In the present study, we tested the hypothesis that social cues from conspecifics modulate electrical activity of TN-GnRH neurons from the intact brain of female medaka fish (Oryzias latipes). We further investigated the potential roles of chemosensory and visual signals in mediating the social cue response. We used a transgenic line of medaka with TN-GnRH neurons genetically tagged with green fluorescent protein, allowing visualization of specific neurons for whole-cell current clamp electrophysiology. We demonstrated that 24-h exposure to male visual and chemosensory cues suppressed the electrical activity of female TN-GnRH neurons compared with exposure to other females. Chemosensory cues alone were insufficient to induce this social cue response. However, visual cues alone replicated the "combined" social cue response. These findings support our hypothesis that sensory signals—and specifically, visual social cues—modulate electrical activity of TN-GnRH neurons.

Vascular endothelial growth factor receptor inhibitor enhances dietary salt-induced hypertension in Sprague-Dawley rats

Gu, J.-W., Manning, R. D., Young, E., Shparago, M., Sartin, B., Bailey, A. P. — 2009-07-02

Clinical evidence links the inhibition of VEGF to hypertension. However, the mechanisms by which VEGF affects the pathogenesis of hypertension remain in question. We determined 1) whether administration of VEGF receptor inhibitor SU5416 enhances dietary salt-induced hypertension in Sprague-Dawley (SD) rats, and 2) whether VEGF or SU5416 directly affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC) and endothelial nitric oxide synthase (eNOS) expression in cultured human glomerular microvessel endothelial cells (HGMEC). Ten 10-wk-old male SD rats received a high sodium diet (HS; 8%) and the other 10 SD rats received a normal sodium diet (NS; 0.5%) for 4 wks. After 2 wks of the dietary program, five rats were administered with SU5416 at 10 mg·kg^–1·day^–1 ip or DMSO (vehicle) for 14 days in HS and NS groups. Mean arterial pressure was significantly higher in rats treated with SU5416, as opposed to those treated with DMSO and fed with HS for 4 wk (157.6 ± 3.9 vs. 125.9 ± 4.3 mmHg, P < 0.01). Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group with SU5416 administration, compared with those in the vehicle HS group. 3H-thymidine incorporation assay showed that SU5416 blocked the actions of both exogenous and endogenous VEGF on the proliferation of HRPTEC. VEGF (10 ng/ml) significantly increased eNOS protein levels by 29% in cultured HGMEC, but its action was completely abolished by SU5416. These results suggest that VEGF receptor inhibition enhances dietary salt-induced hypertension and kidney injury, possibly by direct damage on renal cells and decreasing NO production by eNOS.

Hypotension- and osmotically induced thirst in old Brown Norway rats

Thunhorst, R. L., Beltz, T. G., Johnson, A. K. — 2009-07-02

Compared to young cohorts, old rats drink less water in response to several thirst-inducing stimuli. In these experiments, we characterized water drinking in response to hypotension and cellular dehydration in young (4 mo), middle-aged adult (12 mo) and old (29–30 mo) male Brown Norway rats. We injected the vasodilator, minoxidil as an intravenous bolus in a range of doses (0–20 mg/kg), so that drinking responses could be compared at equivalent reductions of arterial pressure. Old rats had greatly diminished reflex tachycardia and became significantly more hypotensive after minoxidil compared with young and middle-aged rats. When compared at equivalent reductions of arterial pressure, old rats drank one-third as much as middle-aged rats, and one-fifth as much as young rats. In addition, there were age-related deficits in drinking in response to a range of administered loads of sodium (0.15–2 M NaCl, 2 ml/100 g body wt). Urinary excretion of water and sodium in response to the loads was equivalent across ages. Both middle-aged and old rats were less able than young rats to repair their water deficits after sodium loading, attributable almost entirely to their reduced drinking responses compared with young rats. Lastly, age-related declines in drinking appeared to be more severe in response to hypotension than in response to cellular dehydration.

Increased prolyl 4-hydroxylase expression and differential regulation of hypoxia-inducible factors in the aged rat brain

Ndubuizu, O. I., Chavez, J. C., LaManna, J. C. — 2009-07-02

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that mediate the adaptive response of mammalian cells and tissues to changes in tissue oxygenation. In the present study, we show an age-dependent decline in cortical HIF-1 accumulation and activation of HIF target genes in response to hypoxia. This inducible response is significantly attenuated in the cerebral cortex of 18-mo-old Fischer 344 rat yet virtually absent in the cerebral cortex of 24-mo-old Fischer 344 rat. This attenuated HIF-1 response had no effect on mRNA upregulation of HIF-independent genes in the aged cortex. We have provided evidence that this absent HIF-1 response is directly correlated with an increase in the expression of the HIF regulatory enzyme, prolyl 4-hydroxylase (PHD). In addition, our study shows that cortical HIF-2 expression in senescent normoxic controls is also significantly greater than that of younger normoxic controls, despite no difference in HIF-2 mRNA levels. The posttranslational regulation of HIF-2 under normoxic conditions seems to be attenuated in the aged rat brain, which is an in vivo demonstration of differential regulation of HIF-1 and HIF-2.

Sex differences in the resistive and elastic work of breathing during exercise in endurance-trained athletes

Guenette, J. A., Querido, J. S., Eves, N. D., Chua, R., Sheel, A. W. — 2009-07-02

It is not known whether the high total work of breathing (WOB) in exercising women is higher due to differences in the resistive or elastic WOB. Accordingly, the purpose of this study was to determine which factors contribute to the higher total WOB during exercise in women. We performed a comprehensive analysis of previous data from 16 endurance-trained subjects (8 men and 8 women) that underwent a progressive cycle exercise test to exhaustion. Esophageal pressure, lung volumes, and ventilatory parameters were continuously monitored throughout exercise. Modified Campbell diagrams were used to partition the esophageal-pressure volume data into inspiratory and expiratory resistive and elastic components at 50, 75, 100 l/min and maximal ventilations and also at three standardized submaximal work rates (3.0, 3.5, and 4.0 W/kg). The total WOB was also compared between sexes at relative submaximal ventilations (25, 50, and 75% of maximal ventilation). The inspiratory resistive WOB at 50, 75, and 100 l/min was 67, 89, and 109% higher in women, respectively (P < 0.05). The expiratory resistive WOB was 131% higher in women at 75 l/min (P < 0.05) with no differences at 50 or 100 l/min. There were no significant sex differences in the inspiratory or expiratory elastic WOB across any absolute minute ventilation. However, the total WOB was 120, 60, 50, and 45% higher in men at 25, 50, 75, and 100% of maximal exercise ventilation, respectively (P < 0.05). This was due in large part to their much higher tidal volumes and thus higher inspiratory elastic WOB. When standardized for a given work rate to body mass ratio, the total WOB was significantly higher in women at 3.5 W/kg (239 ± 31 vs. 173 ± 12 J/min, P < 0.05) and 4 W/kg (387 ± 53 vs. 243 ± 36 J/min, P < 0.05), and this was due exclusively to a significantly higher inspiratory and expiratory resistive WOB rather than differences in the elastic WOB. The higher total WOB in women at absolute ventilations and for a given work rate to body mass ratio is due to a substantially higher resistive WOB, and this is likely due to smaller female airways relative to males and a breathing pattern that favors a higher breathing frequency.

Effect of acute and chronic caloric restriction and metabolic glucoprivation on spontaneous physical activity in obesity-prone and obesity-resistant rats

Teske, J. A., Kotz, C. M. — 2009-07-02

Caloric restriction (CR) and metabolic glucoprivation affect spontaneous physical activity (SPA), but it's unknown whether these treatments similarly affect SPA in selectively bred obesity-prone (OP) and -resistant (OR) rats. OR rats have greater basal SPA and are more responsive to treatments that modulate SPA, such as orexin A administration. We hypothesized that OR rats would be more sensitive to other treatments modulating SPA. To test this, continuous 24-h SPA was measured before and during acute (24 h) and chronic (8 wk) CR in OR, OP, and Sprague-Dawley rats. Pharmacological glucoprivation was produced by injection of 2-deoxyglucose (2-DG), and SPA was measured 5 h postinjection. Acute CR increased SPA in all groups; however, the effect was dependent on the index of SPA and time interval during the 24-h time period. In contrast to OR rats, chronic CR increased distance traveled, ambulatory episodes, and time spent in ambulation and stereotypy during the time interval preceding anticipation of food in OP and Sprague-Dawley rats. Although the effects of 2-DG treatment on SPA were minimal, OR rats had significantly greater SPA than OP and Sprague-Dawley rats independent of treatment. That chronic CR failed to result in significant changes in SPA in OR rats suggests that these rats may be especially unresponsive to treatments modulating feeding. This insensitivity coupled with elevated basal SPA levels may in part mediate phenotypic traits of lean rats.

Hypothalamic paraventricular nucleus mediates sodium-induced changes in cardiovascular and renal function in conscious sheep

Frithiof, R., Ramchandra, R., Hood, S., May, C., Rundgren, M. — 2009-07-02

The contribution of the paraventricular nucleus of the hypothalamus (PVN) in mediating cardiovascular, renal, hormonal, and sympathetic nerve responses to increased cerebrospinal fluid (CSF) [Na⁺] was investigated in conscious sheep. Intracerebroventricular hypertonic NaCl (0.5 mol/l, 20 µl/min for 60 min) increased arterial blood pressure [AP; +13.4 (sd 2.0) mmHg, P < 0.001] and central venous pressure [CVP; +2.8 (sd 1.3) mmHg, P < 0.001], but did not significantly change heart rate or cardiac output (n = 6). Elevated CSF [Na⁺] also lowered plasma ANG II levels [–3.3 (sd 1.6) pmol/l, P = 0.004] and increased creatinine clearance [+31.5 (sd 32.7) ml/min, P = 0.03] and renal sodium excretion [+9.2 (sd 9.2) mmol/h, P = 0.003]. Lidocaine injection (1 µl, 2%) into the PVN prior to the ICV infusion had no apparent effect per se, but it abolished the AP, CVP, creatinine clearance, and ANG II responses to hypertonic NaCl, as well as reducing the increase in renal sodium excretion (n = 6). Subsequent studies were performed in conscious sheep with chronically implanted electrodes for measurement of renal sympathetic nerve activity (RSNA). The effects of ICV hypertonic NaCl on AP and RSNA were measured before and after PVN-injection of glycine (250 nmol in 500 nl artificial CSF). ICV NaCl increased AP and decreased RSNA (P < 0.001). These effects were significantly reduced by glycine (P = 0.02–0.001, n = 5). Saline injected into the PVN (n = 5) or lidocaine injected outside the PVN (n = 6) had no effect on the response to ICV hypertonic NaCl. These results indicate that the PVN is an important mediator of cerebrally induced homeostatic responses to elevated sodium concentration/hyperosmolality.

Zip3 (Slc39a3) functions in zinc reuptake from the alveolar lumen in lactating mammary gland

Kelleher, S. L., Lopez, V., Lonnerdal, B., Dufner-Beattie, J., Andrews, G. K. — 2009-07-02

The lactating mammary gland is composed of multiple cell types that tightly coordinate the accumulation, production, and secretion of milk components, including essential metals such as zinc (Zn). Our previous studies in animal and cell models implicated the Zn transporter Zip3 (Slc39a3) in mammary gland Zn acquisition. Herein, we investigated this hypothesis directly by utilizing Zip3-null mice. Our data verify that Zip3 is expressed in secretory mammary cells; however, Zip3 does not play a major role in Zn import from the maternal circulation. Importantly, the primary localization of Zip3 was associated with the luminal membrane of the secretory mammary cells. Consistent with this localization, Zn transfer studies using ⁶⁵Zn revealed that Zn retention in the secreted milk pool and milk Zn concentration was higher in Zip3-null compared with wild-type mice. Although total mammary gland Zn concentration was not altered, Zip3-null mice also had altered mammary tissue architecture, increased number of apoptotic cells, and reduced mammary gland weight implicating subtle changes in Zip3-mediated intracellular Zn pools in apoptosis regulation. Taken together, our data indicate that Zip3 does not participate in the acquisition of Zn from maternal circulation for secretion into milk but, in contrast, primarily plays a role in the reuptake and cellular retention of Zn in the mammary gland from the previously secreted milk pool, thus regulating cellular function.

Individual variation in macronutrient regulation measured by proton magnetic resonance spectroscopy of human plasma

2009-07-02

Proton nuclear magnetic resonance (¹H-NMR) spectroscopy of plasma provides a global metabolic profiling method that shows promise for clinical diagnostics. However, cross-sectional studies are complicated by a lack of understanding of intraindividual variation, and this limits experimental design and interpretation of data. The present study determined the diurnal variation detected by ¹H NMR spectroscopy of human plasma. Data reduction methods revealed three time-of-day metabolic patterns, which were associated with morning, afternoon, and night. Major discriminatory regions for these time-of-day patterns included the various kinds of lipid signals (-CH₂- and -CH₂OCOR), and the region between 3 and 4 ppm heavily overlapped with amino acids that had -CH and -CH₂. The phasing and duration of time-of-day patterns were variable among individuals, apparently because of individual difference in food processing/digestion and absorption and clearance of macronutrient energy sources (fat, protein, carbohydrate). The times of day that were most consistent among individuals, and therefore most useful for cross-sectional studies, were fasting morning (0830–0930), postprandial afternoon (1430–1630), and nighttime samples (0430–0530). Importantly, the integrated picture of metabolism provided by ¹H-NMR spectroscopy of plasma suggests that this approach is suitable to study complex regulatory processes, including eating patterns/eating disorders, upper gastrointestinal functions (gastric emptying, pancreatic, biliary functions), and absorption/clearance of macronutrients. Hence, ¹H-NMR spectroscopy of plasma could provide a global metabolic tolerance test to assess complex processes involved in disease, including eating disorders and the range of physiological processes causing dysregulation of energy homeostasis.

The posterior vermis of the cerebellum selectively inhibits 10-Hz sympathetic nerve discharge in anesthetized cats

Barman, S. M., Gebber, G. L. — 2009-07-02

We studied the changes in inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) produced by aspiration or chemical inactivation (muscimol microinjection) of lobule IX (uvula) of the posterior vermis of the cerebellum in baroreceptor-denervated and baroreceptor-innervated cats anesthetized with urethane. Autospectral analysis was used to decompose SND into its frequency components. Special attention was paid to the question of whether the experimental procedures affected the rhythmic (10-Hz and cardiac-related) components of SND. Aspiration or chemical inactivation of lobule IX produced an approximately three-fold increase in the 10-Hz rhythmic component of SND (P ≤ 0.05) in baroreceptor-denervated cats. Total power (0- to 20-Hz band) was unchanged. Despite the absence of a change in total power in SND, there was a statistically significant increase in MAP. In baroreceptor-innervated cats, neither aspiration nor chemical inactivation of the uvula caused a significant change in cardiac-related or total power in SND or MAP. These results are the first to demonstrate a role of cerebellar cortical neurons of the posterior vermis in regulating the frequency composition of naturally occurring SND. Specifically, these neurons selectively inhibit the 10-Hz rhythm-generating network in baroreceptor-denervated, urethane-anesthetized cats. The functional implications of these findings are discussed.

Inflammatory ascites formation induced by macromolecules in mice and rats

Baintner, K. — 2009-07-02

Different macromolecules were administered intraperitoneally to stimulate formation of protein-rich ascitic fluid in rodents. Stimulatory effect of plant lectins depended on the attachment to cell surface carbohydrates, Canavalia ensiformis (ConA) lectin was used in the majority of experiments. The time course of ConA-induced ascites was divided into an early (up to 4 h) and a late (from 6 h on) phase, with a transitional period between the two. Water and protein accumulation showed parallel time courses: volume of the ascitic fluid peaked at around 3 h, and fibrin threads appeared after 6 h. Viscosity of the ascitic fluid and its supernatant increased with time, reaching maximal fibrinogen concentration at around 16 h. Peritoneal permeability, followed by pleural and pericardial effusions, was elicited only by lectins that form soluble complexes with serum glycoproteins, whereas the effect of serum-precipitating lectins was restricted to the peritoneum. Macromolecules with serial positive charges (e.g., polylysine or polyethyleneimine) enhanced peritoneal permeability by ionic interactions with cell surface molecules. Viscosity of the polycation-induced ascitic fluid did not tend to increase with time and corresponded to the early phase of the ConA-induced ascites. Polyglutamate, a polyanionic macromolecule, inhibited the effect of polycations, but not that of ConA. The most efficient stimulatory macromolecules appear to induce ascites by noncovalent cross-linking of cell surface glycoproteins or glycosaminoglycans or both. A similar mechanism may operate in the maintenance of basal secretion to prevent eventual desiccation. Noncovalent cross-linking appears to be a common denominator of both basal and enhanced permeability.

Physiological consequences of gill remodeling in goldfish (Carassius auratus) during exposure to long-term hypoxia

Mitrovic, D., Dymowska, A., Nilsson, G. E., Perry, S. F. — 2009-07-02

Goldfish (Carassius auratus) acclimated to 7°C and exposed to hypoxia (~10 mmHg) for 7 days exhibited a pronounced remodeling of the gill consisting of the removal of an interlamellar cell mass (ILCM). Subsequent experiments were designed to assess the impact of gill remodeling and the associated increase in functional lamellar surface area on the distribution of branchial ionocytes and Cl^– flux across the gill. Despite the increased functional lamellar surface area during hypoxia, there was no corresponding increase in Cl^– loss or efflux of the extracellular marker polyethylene glycol (PEG 4000). However, when hypoxic fish were returned to normoxic water for 12 h, rates of Cl^– and PEG efflux were markedly stimulated in keeping with an increased surface area for solute movement. Similarly, the rate of branchial Cl^– uptake was reduced (105 ± 22 vs. 45 ± 8 µmol·kg^–1·h^–1) in normoxic and hypoxic fish, respectively, but then stimulated (345 µmol·kg^–1·h^–1) upon reestablishment of normoxic conditions. Hypoxia (7 days) was accompanied by a significant decrease in the total cross-sectional area of branchial ionocytes owing to a decrease in their numbers and individual sizes. Thus, despite experiencing an increase in functional lamellar surface area, hypoxic goldfish limit branchial Cl^– loss likely by a hypoxia-mediated decrease in paracellular permeability. In normoxic fish, the ionocytes were largely confined to the outer edges of the ILCM. During hypoxia, preexisting ionocytes migrated with the shrinking ILCM, while a smaller proportion of newly differentiated cells appeared below the surface of the ILCM. The capacity to maintain a population of ionocytes in contact with the water is an appropriate strategy to retain ionoregulatory capabilities regardless of whether the lamellae are uncovered or covered.

Overexpression of follistatin in trout stimulates increased muscling

Medeiros, E. F., Phelps, M. P., Fuentes, F. D., Bradley, T. M. — 2009-07-02

Deletion or inhibition of myostatin in mammals has been demonstrated to markedly increase muscle mass by hyperplasia, hypertrophy, or a combination of both. Despite a remarkably high degree of conservation with the mammalian protein, the function of myostatin remains unknown in fish, many species of which continue muscle growth throughout the lifecycle by hyperplasia. Transgenic rainbow trout (Oncorhynchus mykiss) overexpressing follistatin, one of the more efficacious antagonists of myostatin, were produced to investigate the effect of this protein on muscle development and growth. P₁ transgenics overexpressing follistatin in muscle tissue exhibited increased epaxial and hypaxial muscling similar to that observed in double-muscled cattle and myostatin null mice. The hypaxial muscling generated a phenotype reminiscent of well-developed rectus abdominus and intercostal muscles in humans and was dubbed "six pack." Body conformation of the transgenic animals was markedly altered, as measured by condition factor, and total muscle surface area increased. The increased muscling was due almost exclusively to hyperplasia as evidenced by a higher number of fibers per unit area and increases in the percentage of smaller fibers and the number of total fibers. In several individuals, asymmetrical muscling was observed, but no changes in mobility or behavior of follistatin fish were observed. The findings indicate that overexpression of follistatin in trout, a species with indeterminate growth rate, enhances muscle growth. It remains to be determined whether the double muscling in trout is due to inhibition of myostatin, other growth factors, or both.