The natriuretic peptide system of a euryhaline teleost, the Japanese eel (Anguilla japonica), consists of three types of hormones [atrial natriuretic peptide (ANP), ventricular natriuretic peptide (VNP), and C-type natriuretic peptide (CNP)] and four types of receptors [natriuretic peptide receptors (NPR)-A, -B, -C, and -D]. Although ANP is recognized as a volume-regulating hormone that extrudes both Na⁺ and water in mammals, ANP more specifically extrudes Na⁺ in eels. Accumulating evidence shows that ANP is secreted in response to hypernatremia and acts to inhibit the uptake and to stimulate the excretion of Na⁺ but not water, thereby promoting seawater (SW) adaptation. In fact, ANP is secreted immediately after transfer of eels to SW and ameliorates sudden increases in plasma Na⁺ concentration through inhibition of drinking and intestinal absorption of NaCl. ANP also stimulates the secretion of cortisol, a long-acting hormone for SW adaptation, whereas ANP itself disappears quickly from the circulation. Thus ANP is a primary hormone responsible for the initial phase of SW adaptation. By contrast, CNP appears to be a hormone involved in freshwater (FW) adaptation. Recent data show that the gene expression of CNP and its specific receptor, NPR-B, is much enhanced in FW eels. In fact, CNP infusion increases ²²Na uptake from the environment in FW eels. These results show that ANP and CNP, despite high sequence identity, have opposite effects on salinity adaptation in eels. This difference apparently originates from the difference in their specific receptors, ANP for NPR-A and CNP for NPR-B. VNP may compensate the effects of ANP and CNP for adaptation to respective media, because it has high affinity to both receptors. On the basis of these data, the authors suggest that the natriuretic peptide system is a key endocrine system that allows this euryhaline fish to adapt to diverse osmotic environments, particularly in the initial phase of adaptation.

Hemodynamic studies were performed to determine if blunting of vascular pressor responsiveness to vasoconstrictors during pregnancy may be due to impaired L-type voltage-dependent calcium channels (L-VDCC). Bay K 8644 (BAY), an L-VDCC agonist, was infused in pregnant and nonpregnant anesthetized rabbits (10, 20, 40, and 60 μg/kg) and pregnant and nonpregnant conscious, chronically instrumented (conscious) rabbits (10, 25, and 50 μg/kg). BAY infusions resulted in greater elevation of mean arterial pressure in both anesthetized pregnant (n = 6) vs. nonpregnant (n = 6) (P < 0.05) and conscious pregnant (n = 10) vs. nonpregnant (n = 10) rabbits (P < 0.05). Fractional increase over baseline of total peripheral resistance index was greater in pregnant (36 ± 5 to 78 ± 14%) vs. nonpregnant rabbits (14 ± 4 to 52 ± 6%) (P< 0.02). Cardiac output index did not differ. There was a single high-affinity L-VDCC antagonist aortic binding site with similar number and affinity in pregnant (n = 7) and nonpregnant (n = 7) rabbits. In conclusion, stimulation of L-VDCC induces greater pressor responses in pregnant rabbits with heightened peripheral vasoconstriction. This does not appear to be due to a change in L-VDCC receptor parameters.

To determine whether nitric oxide (NO), adenosine (Ado) receptors, or ATP-sensitive potassium (K_ATP) channels play a role in arteriolar dilations induced by muscle contraction, we used a cremaster preparation in anesthetesized hamsters in which we stimulated four to five muscle fibers lying perpendicular to a transverse arteriole (maximal diameter ∼35–65 μm). The diameter of the arteriole at the site of overlap of the stimulated muscle fibers (the local site) and at a remote site ∼1,000 μm upstream (the upstream site) was measured before, during, and after muscle contraction. Two minutes of 4-Hz muscle stimulation (5–15 V, 0.4 ms) produced local and upstream dilations of 19 ± 1 and 10 ± 1 μm, respectively.N ^ω-nitro-l-arginine (10⁻⁴ M; NO synthase inhibitor), xanthine amine congener (XAC; 10⁻⁶ M; Ado A₁, A_2A, and A_2B receptor antagonist), or glibenclamide (Glib; 10⁻⁵ M; K_ATP channel inhibitor) superfused over the preparation attenuated the local dilation (by 29.7 ± 12.7, 61.8 ± 9.0, and 51.9 ± 14.9%, respectively), but only XAC and Glib attenuated the upstream dilation (by 68.9 ± 6.8 and 89.1 ± 6.4%, respectively). Furthermore, only Glib, when applied to the upstream site directly, attenuated the upstream dilation (48.1 ± 9.1%). Neither XAC nor Glib applied directly to the arteriole between the local and the upstream sites had an effect on the magnitude of the upstream dilation. We conclude that NO, Ado receptors, and K_ATP channels are involved in the local dilation initiated by contracting muscle and that both K_ATP channels and Ado receptor stimulation, but not NO, play a role in the manifestation of the dilation at the upstream site.

Temperature has a strong influence on the excitability and the contractility of the ectothermic heart that can be alleviated in some species by temperature acclimation. The molecular mechanisms involved in the temperature-induced improvement of cardiac contractility and excitability are, however, still poorly known. The present study examines the role of sarcolemmal K⁺ currents from rainbow trout (Oncorhynchus mykiss) cardiac myocytes after thermal acclimation. The two major K⁺ conductances of the rainbow trout cardiac myocytes were identified as the Ba²⁺-sensitive background inward rectifier current (I _K1) and the E-4031-sensitive delayed rectifier current (I _Kr). In atrial cells, the density ofI _K1 is very low and the density ofI _Kr is remarkably high. The opposite is true for ventricular cells. Acclimation to cold (4°C) modified the two K⁺ currents in opposite ways. Acclimation to cold increases the density of I _Kr and depresses the density ofI _K1. These changes in repolarizing K⁺ currents alter the shape of the action potential, which is much shorter in cold-acclimated than warm-acclimated (17°C) trout. These results provide the first concrete evidence that K⁺channels of trout cardiac myocytes are adaptable units that provide means to regulate cardiac excitability and contractility as a function of temperature.

A low-density primary culture of trout ventricular myocytes in serum-free growth medium was established and maintained for up to 10 days at 17°C. The myocytes retained their normal rod shaped morphology, capacitive surface area of the sarcolemma (SL), and contractile quiescence. However, sarcolemmal cation currents changed significantly, some permanently, some transiently, after 8–10 days of culture. TTX-sensitive sodium current (I _Na) and Ba²⁺-sensitive background inward rectifier potassium current (I _K1) were permanently depressed to 24–28% of their control density measured in freshly isolated myocytes. In contrast, L-type calcium current (I _Ca) was only transiently downregulated; after 2–3 days in culture, the density of the current was 32% of the control and recovered to the control value after 8–10 days in culture. The changes in membrane currents were reflected in the shape of the action potential (AP). After 2–3 days in culture, maximal overshoot potential and resting potential were significantly reduced, and the durations of the AP at 50 and 90% repolarization were significantly increased. These changes became significantly more pronounced after 8–10 days of culture, with the exception of AP duration at 50% repolarization level. The shortening of the early plateau phase may reflect an additional change to an outward current, presumably the rapid component of the delayed rectifier (I _Kr). Although the present findings indicate that fish cardiac myocytes can be maintained in serum-free primary culture for at least 10 days at 17°C, some but not all of the electrophysiological characteristics of the myocytes change markedly during culture. The changes in ion currents were not due to loss of sarcolemmal membrane and therefore are likely to represent altered expression of cation currents as an adaptive response to culture conditions.

We tested the hypothesis that a single bout of dynamic exercise produces a postexercise hypotension (PEH) and α₁-adrenergic receptor hyporesponsiveness in spontaneously hypertensive rats (SHR). The postexercise α₁-adrenergic receptor hyporesponsiveness is due to an enhanced buffering of vasoconstriction by nitric oxide. Male (n = 8) and female (n = 5) SHR were instrumented with a Doppler ultrasonic flow probe around the femoral artery. Distal to the flow probe, a microrenathane catheter was inserted into a branch of the femoral artery for the infusion of the α₁-adrenergic receptor agonist phenylephrine (PE). A microrenathane catheter was inserted into the descending aorta via the left common carotid artery for measurements of arterial pressure (AP) and heart rate. Dose-response curves to PE (3.8 × 10⁻³ − 1.98 × 10⁻²μg/kHz) were generated before and after a single bout of dynamic exercise. Postexercise AP was reduced in male (13 ± 3 mmHg) and female SHR (18 ± 7 mmHg). Postexercise vasoconstrictor responses to PE were reduced in males due to an enhanced influence of nitric oxide. However, in females, postexercise vasoconstrictor responses to PE were not altered. Results suggest that nitric oxide- mediated α₁-adrenergic receptor hyporesponsiveness contributes to PEH in male but not female SHR.

We investigated the interplay of neural and hemodynamic mechanisms in postexercise hypotension (PEH) in hypertension. In 15 middle-aged patients with mild essential hypertension, we evaluated blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR), forearm (FVR) and calf vascular resistance (CVR), and autonomic function [by spectral analysis of R-R interval and BP variabilities and spontaneous baroreflex sensitivity (BRS)] before and after maximal exercise. Systolic and diastolic BP, TPR, and CVR were significantly reduced from baseline 60–90 min after exercise. CO, FVR, and HR were unchanged. The low-frequency (LF) component of BP variability increased significantly after exercise, whereas the LF component of R-R interval variability was unchanged. The overall change in BRS was not significant after exercise vs. baseline, although a significant, albeit small, BRS increase occurred in response to hypotensive stimuli. These findings indicate that in hypertensive patients, PEH is mediated mainly by a peripheral vasodilation, which may involve metabolic factors linked to postexercise hyperemia in the active limbs. The vasodilator effect appears to override a concomitant, reflex sympathetic activation selectively directed to the vasculature, possibly aimed to counter excessive BP decreases. The cardiac component of arterial baroreflex is reset during PEH, although the baroreflex mechanisms controlling heart period appear to retain the potential for greater opposition to hypotensive stimuli.

We developed a new model to examine the role of arterial baroreceptors in the long-term control of mean arterial pressure (MAP) in dogs. Baroreceptors in the aortic arch and one carotid sinus were denervated, and catheters were implanted in the descending aorta and common carotid arteries. MAP and carotid sinus pressure (CSP) averaged 104 ± 2 and 102 ± 2 mmHg (means ± 1 SE), respectively, during a 5-day control period. Baroreceptor unloading was induced by ligation of the common carotid artery proximal to the innervated sinus (n = 6 dogs). MAP and CSP averaged 127 ± 7 and 100 ± 3 mmHg, respectively, during the 7-day period of baroreceptor unloading. MAP was significantly elevated (P < 0.01) compared to control, but CSP was unchanged. Heart rate and plasma renin activity increased significantly in response to baroreceptor unloading. Removal of the ligature to restore normal flow through the carotid resulted in normalization of all variables. Ligation of the carotid below a denervated sinus (n = 4) caused a significant decrease in CSP but no systemic hypertension. These results indicate that chronic unloading of carotid baroreceptors can produce neurogenic hypertension and provide strong evidence that arterial baroreceptors are involved in the long-term control of blood pressure.

The purpose of this study was to evaluate the physiological strain index (PSI) for different age groups during exercise-heat stress (EHS). PSI was applied to three different databases. First, from young and middle-age men (21 ± 2 and 46 ± 5 yr, respectively) matched (n = 9 each,P > 0.05) for maximal aerobic power. Subjects were heat acclimated by daily treadmill walking for two 50-min bouts separated by 10-min rest for 10 days in a hot-dry environment [49°C, 20% relative humidity (RH)]. The second database involved a group (n = 8) of young (YA) and a group (n = 7) of older (OA) men (26 ± 1 and 69 ± 1 yr, respectively) who underwent 16 wk of aerobic training and two control groups (n = 7 each) who were matched for age to YA and OA. These four groups performed EHS at 36°C, 40% RH on a cycle ergometer for 60 min at 60% maximal aerobic power before and after training. The third database was obtained from three groups of postmenopausal women and a group of 10 men. Two groups of women (n = 8 each) were undergoing hormone replacement therapy, estrogen or estrogen plus progesterone, and the third group (n = 9) received no hormone replacement. Subjects were over 50 yr and performed the same EHS: exercising at 36°C, 40% RH on a cycle ergometer for 60 min. PSI assessed the strain for all three databases and reported differences were significant at P < 0.05. This index rated the strain in rank order, whereas the postacclimation and posttraining groups were assessed as having less strain than the preacclimation and pretraining groups. Furthermore, middle-aged women on estrogen replacement therapy had less strain than estrogen + progesterone and no hormone therapy. PSI evaluation was extended for men and women of different ages (50–70 yr) during acute EHS, heat acclimation, after aerobic training, and inclusive of women undergoing hormone replacement therapy.

We demonstrated previously that induction of diabetes in rats that were treated chronically with the nitric oxide synthase inhibitorN ^G-nitro-l-arginine methyl ester (l-NAME) causes a severe, progressive increase in mean arterial pressure. This study tested the role of the sympathetic nervous system in that response. Rats were instrumented with chronic artery and vein catheters and assigned randomly to four diabetic groups pretreated with vehicle (D), l-NAME (D+L), the α₁- and β-adrenergic receptor antagonists terazosin and propranolol (D+B), or l-NAME, terazosin, and propranolol (D+LB). After baseline measurements were taken, rats were pretreated; 6 days later, streptozotocin was administered and 3 wk of diabetes ensued. D+L rats had a marked, progressive increase in arterial pressure that by day 20 was ∼60 mmHg greater than in D rats. The pressor response to l-NAME was significantly attenuated in diabetic rats cotreated with adrenergic blockers. Duringweek 1 of diabetes, plasma renin activity (PRA) increased and then returned to control levels in D rats. PRA increased progressively in D+L rats, and chronic adrenergic receptor blockade restored the biphasic renin response in D+LB rats. These results suggest that the sympathetic nervous system may be involved in the hypertensive response to onset of diabetes inl-NAME-treated rats, possibly through control of renin secretion.

Cholecystokinin (CCK) is a potential mediator of gastrointestinal vasodilatation during digestion. To determine whether CCK influences sympathetic vasomotor function, we examined the effect of systemic CCK administration on mean arterial blood pressure (MAP), heart rate (HR), lumbar sympathetic nerve discharge (LSND), splanchnic sympathetic nerve discharge (SSND), and the discharge of presympathetic neurons of the rostral ventrolateral medulla (RVLM) in α-chloralose-anesthetized rats. CCK (1–8 μg/kg iv) reduced MAP, HR, and SSND and transiently increased LSND. Vagotomy abolished the effects of CCK on MAP and SSND as did the CCK-A receptor antagonist devazepide (0.5 mg/kg iv). The bradycardic effect of CCK was unaltered by vagotomy but abolished by devazepide. CCK increased superior mesenteric arterial conductance but did not alter iliac conductance. CCK inhibited a subpopulation (∼49%) of RVLM presympathetic neurons whereas ∼28% of neurons tested were activated by CCK. The effects of CCK on RVLM neuronal discharge were blocked by devazepide. RVLM neurons inhibited by exogenous CCK acting via CCK-A receptors on vagal afferents may control sympathetic vasomotor outflow to the gastrointestinal tract vasculature.

The reduced pressure response to vasopressin during acute sepsis has directed our interest to the regulation of vasopressin V_1A receptors. Rats were injected with lipopolysaccharide for induction of experimental gram-negative sepsis. V_1A receptor gene expression was downregulated in the liver, lung, kidney, and heart during endotoxemia. Inasmuch as the concentrations of proinflammatory cytokines such as interleukin-1β, tumor necrosis factor-α, and interferon-γ were highly increased during sepsis, the influence of these cytokines on V_1A receptor expression was investigated in primary cultures of hepatocytes and in the aortic vascular smooth muscle cell line A7r5. V_1A receptor expression was downregulated by the cytokines in a nitric oxide-independent manner. Blood pressure dose-response studies after injection of endotoxin showed a diminished responsiveness to the selective V₁ receptor agonist Phe²,Ile³,Orn⁸-vasopressin. Our data show that sepsis causes a downregulation of V_1Areceptors and suggest that this effect is likely mediated by proinflammatory cytokines. We propose that this downregulation of V_1A receptors contributes to the attenuated responsiveness of blood pressure in response to vasopressin and, therefore, contributes to the circulatory failure in septic shock.

In previous studies, the heat shock response, induced by hyperthermia or sodium arsenite, increased interleukin (IL)-6 production in intestinal mucosa and cultured human enterocytes. A novel way to induce the heat shock response, documented in other cell types, is treatment with proteasome inhibitors. It is not known if proteasome inhibition induces heat shock in enterocytes or influences IL-6 production. Here we tested the hypothesis that treatment of cultured Caco-2 cells, a human intestinal epithelial cell line, with proteasome inhibitors induces the heat shock response and stimulates IL-6 production. Treatment of Caco-2 cells with one of the proteasome inhibitors MG-132 or lactacystin activated the transcription factor heat shock factors (HSF)-1 and -2 and upregulated cellular levels of the 72-kDa heat shock protein HSP-72. The same treatment resulted in increased gene and protein expression of IL-6, a response that was blocked by quercetin. Additional experiments revealed that the IL-6 gene promoter contains a HSF-responsive element and that the IL-6 gene may be regulated by the heat shock response. The present results suggest that proteasome inhibition induces heat shock response and IL-6 production in enterocytes and that IL-6 may be a heat shock-responsive gene, at least under certain circumstances. The observations are important considering the multiple biological roles of IL-6, both locally in the gut mucosa and systemically, and considering recent proposals in the literature to use proteasome inhibitors in the clinical setting to induce the heat shock response.

Golden-mantled ground squirrels (Spermophilus lateralis) undergo seasonal hibernation during which core body temperature (T_b) values are maintained 1–2°C above ambient temperature. Hibernation is not continuous. Squirrels arouse at ∼7-day intervals, during which T_bincreases to 37°C for ∼16 h; thereafter, they return to hibernation and sustain low T_bs until the next arousal. Over the course of the hibernation season, arousals consume 60–80% of a squirrel's winter energy budget, but their functional significance is unknown and disputed. Host-defense mechanisms appear to be downregulated during the hibernation season and preclude normal immune responses. These experiments assessed immune function during hibernation and subsequent periodic arousals. The acute-phase response to bacterial lipopolysaccharide (LPS) was arrested during hibernation and fully restored on arousal to normothermia. LPS injection (ip) resulted in a 1–1.5°C fever in normothermic animals that was sustained for >8 h. LPS was without effect in hibernating squirrels, neither inducing fever nor provoking arousal, but a fever did develop several days later, when squirrels next aroused from hibernation; the duration of this arousal was increased sixfold above baseline values. Intracerebroventricular infusions of prostaglandin E₂provoked arousal from hibernation and induced fever, suggesting that neural signaling pathways that mediate febrile responses are functional during hibernation. Periodic arousals may activate a dormant immune system, which can then combat pathogens that may have been introduced immediately before or during hibernation.

This study investigated the capacity of conjugated linoleic acids (CLA) to reduce ex vivo antigen-induced release of eicosanoids in a type I hypersensitivity model. Guinea pigs were fed a diet containing 0.25% safflower oil (control) or 0.25% CLA [43% trans(t)10, cis (c)12; 41% c9,t11/t9, c11 18:2] for 2 wk before and during sensitization to ovalbumin (OVA). Lungs, tracheas, and bladders were incubated in physiological saline solution (PSS) for 1 h (basal mediator release) and challenged with OVA (0.01 g/l PSS) for 1 h (mediator release in response to antigen). Eicosanoids were quantified by HPLC/tandem mass spectrometry or enzyme immunoassay. CLA feeding resulted in no change in basal release but decreased eicosanoid release from sensitized tissues in response to antigen challenge in the following manner: thromboxane B₂, 6-keto-prostaglandin (PG)F_1α, PGF_2α, PGD₂, PGE₂ by 57–75% in lung, 45–65% in trachea, and 38–60% in bladder; and leukotriene C₄/D₄/E₄ by 87, 90, and 50% in lung, trachea, and bladder, respectively. These data indicate that feeding CLA reduces lipid-derived inflammatory mediators produced by this type I hypersensitivity model.

The effect of brown adipose tissue (BAT) sympathetic hemidenervation on the activity of glycerokinase (GyK) was investigated in different physiological conditions. In rats fed a balanced diet, the activity of the enzyme was ∼50% lower in BAT-denervated pads than in intact, innervated pads. In rats adapted to a high-protein, carbohydrate-free diet, norepinephrine turnover rates and BAT GyK activity were already reduced, and BAT denervation resulted in a further decrease in the activity of the enzyme. Cold acclimation of normally fed rats at 4°C for 10 days markedly increased the activity of the enzyme. Cold exposure (4°C) for 6 h was insufficient to stimulate BAT GyK, but the activity of the enzyme was already increased after 12 h of cold exposure. The cold-induced BAT GyK stimulation was completely blocked in BAT-denervated pads. The data indicate that an adequate sympathetic flow to BAT is required for the maintenance of normal levels of GyK activity and for the enzyme response to situations, such as cold exposure, which markedly increase BAT sympathetic flow.

To study the role of L-selectin in neutrophil (PMN) margination and sequestration in the pulmonary microcirculation, maximally active concentrations of C5a (900 pmol/g) andN-formylmethionyl-leucyl-phenylalanine (fMLP; 0.34 pmol/g) were injected into the jugular vein of wild-type or L-selectin-deficient C57BL/6 mice. In wild-type mice administered C5a or fMLP, 92 ± 1% and 34 ± 9%, respectively, of peripheral blood PMN were trapped mostly in the pulmonary circulation as determined by immunohistochemistry and myeloperoxidase activity. In wild-type mice treated with F(ab′)₂ fragments of the L-selectin monoclonal antibody MEL-14 or in L-selectin-deficient mice, C5a-induced neutropenia was not significantly reduced, but the decrease in peripheral PMN in response to fMLP was completely abolished, indicating that L-selectin is necessary for fMLP- but not C5a-induced pulmonary margination. Immunostained lung sections of fMLP- or C5a-treated mice showed sequestered neutrophils in alveolar capillaries with no evidence of neutrophil aggregates. We conclude that chemoattractant-induced PMN margination in the pulmonary circulation can occur by two separate mechanisms, one of which requires L-selectin.

Behavioral tests, tightrope success, and exploratory activity in a T maze were conducted with male and female mice for 65 wk. Four groups were defined: the lower performance slow males and slow females and the higher performance fast males and fast females. Fast females showed the longest life span and the highest performance, and slow males showed the lowest performance and the shortest life span. Oxidative stress and mitochondrial electron transfer activities were determined in brain of young (28 wk), adult (52 wk), and old (72 wk) mice in a cross-sectional study. Brain thiobarbituric acid reactive substances (TBARS) were increased by 50% in old mice and were ∼15% higher in males than in females and in slow than in fast mice. Brain Cu,Zn-superoxide dismutase (SOD) activity was increased by 52% and Mn-SOD by 108% in old mice. The activities of mitochondrial enzymes NADH-cytochrome c reductase, cytochrome oxidase, and citrate synthase were decreased by 14–58% in old animals. The cumulative toxic effects of oxyradicals are considered the molecular mechanism of the behavioral deficits observed on aging.

To establish physiological mechanisms for fetal growth restriction in pregnant adolescent ewes we studied uterine, fetal, and uteroplacental metabolism in ewes offered a high (n = 12) or moderate (n = 10) dietary intake. High intakes decreased placental (226 vs. 414 g, P < 0.001) and fetal weight (3,323 vs. 4,626 g, P < 0.01). Uterine blood flow was reduced absolutely (−36%) but proportional to conceptus weight; umbilical blood flow was reduced absolutely (−37%) and per fetal weight (−15%). Uterine oxygen uptake was decreased per conceptus weight (−14%); there was no change in fetal weight oxygen consumption. Uteroplacental oxygen consumption and clearance were reduced proportional to weight. Similar changes were measured for glucose fluxes and fetal glucose concentration; fetal insulin concentration was reduced. In this model of fetal growth restriction, therefore, maintenance of fetal weight-specific glucose and oxygen consumption rates are producing relative hypoglycemia and hypoxemia. This indicates that increased fetal glucose clearance and/or insulin sensitivity may be operating as compensatory mechanisms to preserve normal fetal metabolism while fetal growth is sacrificed.

The aim of this study was to determine whether extracellular ATP ([ATP]_o) stimulated a Ca²⁺-activated K⁺ efflux in trophoblast cells that was dependent on extracellular Ca²⁺([Ca²⁺]_o). Cytotrophoblast cells, isolated from human placenta, were examined following 18 h (relatively undifferentiated) and 66 h (multinucleate cells) of culture. Potassium efflux was measured using ⁸⁶Rb as a trace marker. Intracellular Ca²⁺ ([Ca²⁺]_i) was examined by microfluorometry using fura 2. [ATP]_osignificantly increased ⁸⁶Rb efflux to a peak that declined to control (18-h cells) or an elevated plateau (66-h cells) and was inhibited by 100 nM charybdotoxin. Removing [Ca²⁺]_o significantly reduced⁸⁶Rb efflux in both groups as did application of 150 μM GdCl₃. [ATP]_o significantly increased [Ca²⁺]_i in both groups of cells. The response was reduced by removing [Ca²⁺]_o and applying 150 μM GdCl₃. For both ⁸⁶Rb efflux and microfluorometry experiments, the response to [ATP]_o was more dependent on [Ca²⁺]_o in 66-h cells compared with 18-h cells (∼70% greater). Cytotrophoblast cells exhibit an [ATP]_o-stimulated Ca²⁺-activated K⁺ efflux. The dependency of this pathway on [Ca²⁺]_o is greater in the 66-h multinucleate syncytiotrophoblast-like cells, suggesting that the mechanism for Ca²⁺ entry may be altered during differentiation of trophoblast cells.

Rearing animals in small litters induces a permanent increase in body weight and body fat. To determine whether changes in sympathoadrenal activity contribute to this effect, litter size was adjusted the day after birth and maintained until weaning at 21 days. Sympathetic nervous system (SNS) activity was measured in adult animals using [³H]norepinephrine ([³H]NE) turnover in peripheral tissues. Although litter size was without effect on [³H]NE turnover in chow-fed animals, acceleration of [³H]NE turnover by dietary sucrose was completely abolished in heart and attenuated in interscapular brown adipose tissue and kidney of rats reared in small litters. Body and epididymal fat-pad weights were heavier in rats reared in small litters; however, weight gain in response to dietary enrichment with sucrose did not differ as a function of litter size. Thus litter size alters dietary activation of the SNS, and this effect presumably reflects changes in central nervous system regulation.

ANG II is capable of stimulating expression of immediate early genes such as egr-1 and c-fos in a variety of cultured cells, including cells of renal origin. To investigate whether ANG II can stimulate early growth response gene expression in vivo, we studied the effects of acute renal artery infusion of low-dose ANG II (2.5 ng · kg⁻¹ · min⁻¹) or vehicle on the renal expression of c-fos and egr-1 genes in rats. ANG II infusion for 30 or 240 min decreased renal vascular conductance by ∼13 and 8%, respectively, compared with the vehicle group. Expression of the early growth response genes c-fosand egr-1 was analyzed using Northern blot hybridization. No significant upregulation of c-fos or egr-1 mRNA levels was detected in rats that received ANG II for either 30 or 240 min, compared with the vehicle groups. We conclude that ANG II, at doses that cause significant physiological effects, does not increase the renal expression of c-fos or egr-1 genes over periods of up to 4 h in vivo.

The present study was designed to examine whether changes in Ca²⁺ release by inositol-1,4,5-trisphosphate (IP₃) in 8-, 15-, and 30-day-old rat skeletal muscles could be associated with the expression of IP₃ receptors. Experiments were conducted in slow-twitch muscle in which both IP₃-induced Ca²⁺ release and IP₃-receptor (IP₃R) expression have been shown to be larger than in fast-twitch muscle. In saponin-skinned fibers, IP₃ induced transient contractile responses in which the amplitude was dependent on the Ca²⁺-loading period with the maximal IP₃ contracture being at 20 min of loading. The IP₃ tension decreased during postnatal development, was partially inhibited by ryanodine (100 μM), and was blocked by heparin (20–400 μg/ml). Amplification of the DNA sequence encoding for IP₃R isoforms (using the RT-PCR technique) showed that in slow-twitch muscle, the type 2 isoform is mainly expressed, and its level decreases during postnatal development in parallel with changes in IP₃ responses in immature fibers. IP₃-induced Ca²⁺ release would then have greater participation in excitation-contraction coupling in developing fibers than in mature muscle.

During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5′-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548–551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870–R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol202: 2787–2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052–3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188–R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787–2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996). This leads via erythroid β-adrenergic/adenosine A₂ receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al.,Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon et al., Blood 91: 3052–3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973–R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP might promote general differentiating processes in erythroid cells, thereby allowing adaptive modulation of the latest steps of erythroid differentiation during developmental hypoxia.

Dendroaspis natriuretic peptide (DNP) is a recently discovered peptide with structural similarity to known natriuretic peptides. DNP has been shown to possess potent renal actions. Our objectives were to define the acute hemodynamic actions of DNP in normal anesthetized dogs and the acute effects of DNP on left ventricular (LV) function in conscious chronically instrumented dogs. In anesthetized dogs, DNP, but not placebo, decreased mean arterial pressure (141 ± 6 to 109 ± 7 mmHg, P < 0.05) and pulmonary capillary wedge pressure (5.8 ± 0.3 to 3.4 ± 0.2 mmHg, P < 0.05). Cardiac output decreased and systemic vascular resistance increased with DNP and placebo. DNP-like immunoreactivity and guanosine 3′,5′-cyclic monophosphate concentration increased without changes in other natriuretic peptides. In conscious dogs, DNP decreased LV end-systolic pressure (120 ± 7 to 102 ± 6 mmHg,P < 0.05) and volume (32 ± 6 to 28 ± 6 ml,P < 0.05) and LV end-diastolic volume (38 ± 5 to 31 ± 4 ml, P < 0.05) but not arterial elastance. LV end-systolic elastance increased (6.1 ± 0.7 to 7.4 ± 0.6 mmHg/ml, P < 0.05), and Tau decreased (31 ± 2 to 27 ± 1 ms, P < 0.05). The effects on hemodynamics, LV function, and second messenger generation suggest synthetic DNP may have a role as a cardiac unloading and lusitropic peptide.

In an unanesthetized decerebrate in situ arterially perfused brain stem preparation of mature rat, strychnine (0.05-0.2 μM) blockade of glycine receptors caused postinspiratory glottal constriction to occur earlier, shifting from early expiration to inspiration. This resulted in a paradoxical inspiratory-related narrowing of the upper airway. Stimulation of the trigeminal ethmoidal nerve (EN5; 20 Hz, 100 μs, 0.5–2 V) evoked a diving response, which included a reflex apnea, glottal constriction, and bradycardia. After strychnine administration, this pattern was converted to a maintained phrenic nerve discharge and a reduced glottal constriction that was interrupted intermittently by transient abductions. The onset of firing of postinspiratory neurons shifted from early expiration into neural inspiration in the presence of strychnine, but neurons maintained their tonic activation during EN5 stimulation, as observed during control. Inspiratory neurons that were hyperpolarized by EN5 stimulation in control conditions were powerfully excited after loss of glycinergic inhibition. Thus the integrity of glycinergic inhibition within the pontomedullary respiratory network is critical for the coordination of cranial and spinal motor outflows during eupnea but also for protective reflex regulation of the upper airway.

One characteristic of heart failure (HF) is increased sympathetic activation. The paraventricular nucleus (PVN) of the hypothalamus (involved in control of sympathetic outflow) has been shown to have increased neuronal activation during HF. This study examined the influence of endogenous GABA input (inhibitory in nature) into the PVN on renal sympathetic nerve discharge (RSND), arterial blood pressure (BP), and heart rate (HR) in rats with HF induced by coronary artery ligation. In α-chloralose- and urethane-anesthetized rats, microinjection of bicuculline (a GABA antagonist) into the PVN produced a dose-dependent increase in RSND, BP, and HR in both sham-operated control and HF rats. Bicuculline attenuated the increase in RSND and BP in HF rats compared with control rats. Alternatively, microinjection of the GABA agonist muscimol produced a dose-dependent decrease in RSND, BP, and HR in both control and HF rats. Muscimol was also less effective in decreasing RSND, BP, and HR in HF rats than in control rats. These results suggest that endogenous GABA-mediated input into the PVN of rats with HF is less effective in suppressing RSND and BP compared with control rats. This is partly due to the post-release actions of GABA, possibly caused by altered function of post-synaptic GABA receptors in the PVN of rats with HF. Reduced GABA-mediated inhibition in the PVN may contribute to increased sympathetic outflow, which is commonly observed during HF.

In response to an acute hypoxemic insult, the mammalian fetus shows a redistribution of the cardiac output in favor of the heart and brain. Peripheral vasoconstriction contributes to this response and is partly mediated by the release of catecholamines. Two mechanisms of catecholamine release in the fetus are reported: 1) neurogenic sympathetic stimulation and2) a nonneurogenic mechanism via a direct effect of hypoxemia on chromaffin tissues. In the present study, the effects of sympathetic blockade on plasma catecholamine release and cardiac output distribution in response to acute hypoxemia were studied in the chick embryo at different stages of incubation. Only at the end of the incubation period, sympathetic blockade markedly attenuated the increase in plasma catecholamine concentrations and resulted in a greater fraction of the cardiac output distributed to the carcass. However, these effects did not prevent a significant increase in cardiac output to the brain and heart during acute hypoxemia. These data imply that in the chick embryo the contribution of neurogenic mechanisms to the catecholaminergic response to acute hypoxemia becomes greater by the end of the incubation period.

We investigated circadian and homeostatic regulation of nonrapid eye movement (NREM) sleep in golden-mantled ground squirrels during euthermic intervals between torpor bouts. Slow-wave activity (SWA; 1–4 Hz) and sigma activity (10–15 Hz) represent the two dominant electroencephalographic (EEG) frequency components of NREM sleep. EEG sigma activity has a strong circadian component in addition to a sleep homeostatic component, whereas SWA mainly reflects sleep homeostasis [Dijk DJ and Czeisler CA. J Neurosci 15: 3526–3538, 1995; Dijk DJ, Shanahan TL, Duffy JF, Ronda JM, and Czeisler CA.J Physiol (Lond) 505: 851–858, 1997]. Animals maintained under constant conditions continued to display circadian rhythms in both sigma activity and brain temperature throughout euthermic intervals, whereas sleep and wakefulness showed no circadian organization. Instead, sleep and wakefulness were distributed according to a 6-h ultradian rhythm. SWA, NREM sleep bout length, and sigma activity responded homeostatically to the ultradian sleep-wake pattern. We suggest that the loss of sleep-wake consolidation in ground squirrels during the hibernation season may be related to the greatly decreased locomotor activity during the hibernation season and may be necessary for maintenance of multiday torpor bouts characteristic of hibernating species.

The goals of this study were first to determine the effect of temperature on the force loss that results from eccentric contractions in mouse extensor digitorum longus (EDL) muscles and then to evaluate a potential role for altered Ca²⁺ homeostasis explaining the greater isometric force loss observed at the higher temperatures. Isolated muscles performed five eccentric or five isometric contractions at either 15, 20, 25, 30, 33.5, or 37°C. Isometric force loss, caffeine-induced force, lactate dehydrogenase (LDH) release, muscle accumulation of ⁴⁵Ca²⁺ from the bathing medium, sarcoplasmic reticulum (SR) Ca²⁺ uptake, and resting muscle fiber free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured. The isometric force loss after eccentric contractions increased progressively as temperature rose; at 15°C, there was no significant loss of force, but at 37°C, there was a 30–39% loss of force. After eccentric contractions, caffeine-induced force was not affected by temperature nor was it different from that of control muscles at any temperature. Loss of cell membrane integrity and subsequent influx of extracellular Ca²⁺ as indicated by LDH release and muscle⁴⁵Ca²⁺ accumulation, respectively, were minimal over the 15–25°C range, but both increased as an exponential function of temperature between 30 and 37°C. SR Ca²⁺uptake showed no impairment as temperature increased, and the eccentric contraction-induced rise in resting fiber [Ca²⁺]_i was unaffected by temperature over the 15–25°C range. In conclusion, the isometric force loss after eccentric contractions is temperature dependent, but the temperature dependency does not appear to be readily explainable by alterations in Ca²⁺ homeostasis.

The glomerular filtration rate (GFR) normally increases during glycine infusion, which is a test of “renal reserve.” Renal reserve is absent in diabetes mellitus. GFR increases after protein feeding because of increased tubular reabsorption, which reduces the signal for tubuloglomerular feedback (TGF). Dietary protein restriction normalizes some aspects of glomerular function in diabetes. Renal micropuncture was performed in rats 4–5 wk after diabetes was induced by streptozotocin to determine whether renal reserve is lost as a result of altered tubular function and activation of TGF, whether 10 days of dietary protein restriction could restore renal reserve, and whether this results from effects of glycine on the tubule. TGF activation was determined by locating single-nephron GFR (SNGFR) in the early distal tubule along the TGF curve. The TGF signal was determined from the ionic content of the early distal tubule. In nondiabetic rats, SNGFR in the early distal tubule increased during glycine infusion because of primary vasodilation augmented by increased tubular reabsorption, which stabilized the TGF signal. In diabetic rats, glycine reduced reabsorption, thereby activating TGF, which was largely responsible for the lack of renal reserve. In protein-restricted diabetic rats, the tubular response to glycine remained abnormal, but renal reserve was restored by a vascular mechanism. Glycine affects GFR directly and via the tubule. In diabetes, reduced tubular reabsorption dominates. In low-protein diabetes, the vascular effect is enhanced and overrides the effect of reduced tubular reabsorption.

The responses to AT₁-receptor blockade (candesartan 1 mg/kg) and to concomitant volume expansion (saline 35 ml/kg for 90 min) with and without nitric oxide synthase (NOS) inhibition (N ^G-nitro-l-arginine methyl ester 30 μg · kg⁻¹ · min⁻¹) were investigated in separate experiments in normal dogs. AT₁ blockade decreased arterial pressure (106 ± 4 to 96 ± 5 mmHg) and increased glomerular filtration rate (GFR) by 17% and sodium excretion threefold. NOS inhibition increased arterial pressure (103 ± 3 to 116 ± 3 mmHg) and decreased GFR by 21% and reduced sodium excretion by some 80%. Volume expansion increased arterial pressure significantly in all series involving this procedure, most pronounced during combined AT₁ blockade and NOS inhibition (21 ± 4 mmHg). Volume expansion during AT₁ blockade elicited marked natriuresis (26 ± 11 to 274 ± 55 μmol/min) that was severely reduced by concomitant NOS inhibition (10 ± 3 to 45 ± 11 μmol/min), but still much larger than that seen with volume expansion during NOS inhibition alone (2 ± 1 to 23 ± 7 μmol/min). Volume expansion during AT₁ blockade increased GFR (+30%), less so during combined AT₁ blockade and NOS inhibition (+13%), but it did not increase GFR significantly (P = 0.07) during NOS inhibition alone. Plasma ANG II increased greater than sevenfold with AT₁ blockade and doubled with NOS inhibition (pairedt-test, P < 0.05), whereas it decreased by 50–80% during volume expansion irrespective of pretreatment, i.e., during NOS inhibition, volume expansion did not generate subnormal plasma ANG II concentrations. In conclusion, 1) acute AT₁ blockade leads to hyperfiltration, natriuresis, and hyperresponsiveness to volume expansion, 2) these responses are >85% inhibitable by unspecific NOS inhibition, and 3) NOS inhibition alone is followed by increases in plasma ANG II, hypofiltration, and severe antinatriuresis that may be counterbalanced but not overwhelmed by volume expansion. Thus NOS inhibition virtually abolishes the volume expansion natriuresis, at least in part, due to the lack of appropriate inhibition of the renin-angiotensin-aldosterone system.

The responses to infusion of nitric oxide synthase substrate (l-arginine 3 mg · kg⁻¹ · min⁻¹) and to slow volume expansion (saline 35 ml/kg for 90 min) alone and in combination were investigated in separate experiments. l-Arginine left blood pressure and plasma ANG II unaffected but decreased heart rate (6 ± 2 beats/min) and urine osmolality, increased glomerular filtration rate (GFR) transiently, and caused sustained increases in sodium excretion (fourfold) and urine flow (0.2 ± 0.0 to 0.7 ± 0.1 ml/min). Volume expansion increased arterial blood pressure (102 ± 3 to 114 ± 3 mmHg), elevated GFR persistently by 24%, and enhanced sodium excretion to a peak of 251 ± 31 μmol/min, together with marked increases in urine flow, osmolar and free water clearances, whereas plasma ANG II decreased (8.1 ± 1.7 to 1.6 ± 0.3 pg/ml). Combined volume expansion and l-arginine infusion tended to increase arterial blood pressure and increased GFR by 31%, whereas peak sodium excretion was enhanced to 335 ± 23 μmol/min at plasma ANG II levels of 3.0 ± 1.1 pg/ml; urine flow and osmolar clearance were increased at constant free water clearance. In conclusion, l-arginine 1) increases sodium excretion, 2) decreases basal urine osmolality,3) exaggerates the natriuretic response to volume expansion by an average of 50% without persistent changes in GFR, and4) abolishes the increase in free water clearance normally occurring during volume expansion. Thus l-arginine is a natriuretic substance compatible with a role of nitric oxide in sodium homeostasis, possibly by offsetting/shifting the renal response to sodium excess.

Intraventricular injections of vasopressin (VP) and antagonists with varying degrees of specificity for the VP receptors were used to identify the action of endogenous brain VP on 0.3 M NaCl intake by sodium-deficient rats. Lateral ventricular injections of 100 ng and 1 μg VP caused barrel rotations and a dramatic decrease in NaCl intake by sodium-deficient rats and suppressed sucrose intake. Intraventricular injection of the V₁/V₂ receptor antagonist [d(CH₂)₅ ¹,O-Et-Tyr²,Val⁴, Arg⁸]VP and the V₁ receptor antagonist [d(CH₂)₅ ¹,O-Me-Tyr²,Arg⁸]VP (MeT-AVP) significantly suppressed NaCl intake by sodium-deficient rats without causing motor disturbances. MeT-AVP had no effect on sucrose intake (0.1 M). In contrast, the selective V₂ receptor antagonist had no significant effect on NaCl intake. Last, injections of 100 ng MeT-AVP decreased mean arterial blood pressure (MAP), whereas 100 ng VP elevated MAP and pretreatment with MeT-AVP blocked the pressor effect of VP. These results indicate that the effects produced by 100 ng MeT-AVP represent receptor antagonistic activity. These findings suggest that the effect of exogenous VP on salt intake is secondary to motor disruptions and that endogenous brain VP neurotransmission acting at V₁ receptors plays a role in the arousal of salt appetite.

To determine the effects of brief food restriction on fatty acid (FA) metabolism, hindlimbs of F344/BN rats fed either ad libitum (AL) or food restricted (FR) to 60% of baseline food intake for 28 days were perfused under hyperglycemic-hyperinsulinemic conditions (20 mM glucose, 1 mM palmitate, 1,000 μU/ml insulin, [3-³H]glucose, and [1-¹⁴C]palmitate). Basal glucose and insulin levels were significantly lower (P < 0.05) in FR vs. AL rats. Palmitate uptake (34.3 ± 2.7 vs. 24.5 ± 3.1 nmol/g/min) and oxidation (3.8 ± 0.2 vs. 2.7 ± 0.3 nmol · g⁻¹ · min⁻¹) were significantly higher (P < 0.05) in FR vs. AL rats, respectively. Glucose uptake was increased in FR rats and was accompanied by significant increases in red and white gastrocnemius glycogen synthesis, indicating an improvement in insulin sensitivity. Although muscle triglyceride (TG) levels were not significantly different between groups, glucose uptake and total preperfusion TG concentration were negatively correlated (r ² = 0.27, P < 0.05). In conclusion, our results show that under hyperglycemic-hyperinsulinemic conditions, brief FR resulted in an increase in FA oxidative disposal that may contribute to the improvement in insulin sensitivity.

Peripheral and hypothalamic mechanisms underlying the hyperphagia of lactation have been investigated in sheep. Sheep were fed ad libitum and killed at 6 and 18 days of lactation; ad libitum-fed nonlactating sheep were killed as controls. Despite increased food intake, lactating ewes were in negative energy balance. Lactation decreased plasma leptin and adipose tissue leptin mRNA concentrations. OB-Rb gene expression, determined by in situ hybridization, was increased in the hypothalamic arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) at both stages of lactation. Neuropeptide Y (NPY) was increased by lactation in both the ARC and dorsomedial hypothalamus (DMH), although increased gene expression in the DMH was only apparent at day 18 of lactation. Gene expression was decreased for cocaine- and amphetamine-regulated transcript (CART) in the ARC and VMH and for proopiomelanocortin in ARC during lactation. Agouti-related peptide gene expression was increased in the ARC, and melanocortin receptor expression was unchanged in both the ARC and VMH with lactation. Thus the hypoleptinemia of lactation may activate NPY orexigenic pathways and attenuate anorexigenic melanocortin and CART pathways in the hypothalamus to promote the hyperphagia of lactation.