Most previous stress-immune research focused on the immunosuppressive effects of stress on acquired immunity. More recently, it has become clear that acute stressor exposure can potentiate innate, as well as suppress acquired, immunity. For example, acute stress improves recovery from bacterial inflammation, a classic in vivo measure of innate immunity. The previous work was done in sedentary organisms. Physical activity status can modulate the impact of stress on immune function. The following studies tested the hypothesis that the effect of stress on inflammation after subcutaneous challenge with bacteria (Escherichia coli) is facilitated by physical activity. The results were that sedentary, stressed rats resolved their inflammation 1–2 days faster and have increased circulating neutrophils compared with their nonstressed, sedentary counterparts. In contrast, physically active, stressed rats resolve their inflammation 3–4 days faster and have increased circulating and inflammatory site neutrophils compared with their nonstressed counterparts. Importantly, the beneficial impact of stress on inflammation recovery and neutrophil migration was greater in the physically active, than sedentary, stressed rats. Thus physical activity status facilitates the positive effect of acute stress on innate immunity.

Bacterial lipopolysaccharide (LPS) induces fever that is mediated by pyrogenic cytokines such as interleukin (IL)-1β. We hypothesized that the anti-inflammatory cytokine IL-10 modulates the febrile response to LPS by suppressing the production of pyrogenic cytokines. In rats, intravenous but not intracerebroventricular infusion of IL-10 was found to attenuate fever induced by peripheral administration of LPS (10 μg/kg iv). IL-10 also suppressed LPS-induced IL-1β production in peripheral tissues and in the brain stem. In contrast, central administration of IL-10 attenuated the febrile response to central LPS (60 ng/rat icv) and decreased IL-1β production in the hypothalamus and brain stem but not in peripheral tissues and plasma. Furthermore, intravenous LPS upregulated expression of IL-10 receptor (IL-10R1) mRNA in the liver, whereas intracerebroventricular LPS enhanced IL-10R1 mRNA in the hypothalamus. We conclude that IL-10 modulates the febrile response by acting in the periphery or in the brain dependent on the primary site of inflammation and that its mechanism of action most likely involves inhibition of local IL-1β production.

Central neuropeptides play important roles in many physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Central injection of corticotropin-releasing factor (CRF) aggravates carbon tetrachloride (CCl4)-induced acute liver injury through the sympathetic nervous pathway in rats. However, still nothing is known about a role of endogenous neuropeptides in the brain in hepatic pathophysiological regulations. Involvement of endogenous CRF in the brain in CCl4-induced acute liver injury was investigated by centrally injecting a CRF receptor antagonist in rats. Male fasted Wistar rats were injected with CRF receptor antagonist α-helical CRF-(9–41) (0.125–5 μg) intracisternally just before and 6 h after CCl4 (2 ml/kg) administration, and blood samples were obtained before and 24 h after CCl4 injection for measurement of hepatic enzymes. The liver sample was removed 24 h after CCl4 injection, and histological changes were examined. Intracisternal α-helical CRF-(9–41) dose dependently (0.25–2 μg) reduced the elevation of alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal α-helical CRF-(9–41) reduced CCl4-induced liver histological changes, such as centrilobular necrosis. The effect of central CRF receptor antagonist on CCl4-induced liver injury was abolished by sympathectomy and 6-hydroxydopamine pretreatment but not by hepatic branch vagotomy or atropine pretreatment. These findings suggest the regulatory role of endogenous CRF in the brain in experimental liver injury in rats.

Conjugated linoleic acid (CLA) is reported to have health benefits, including reduction of body fat. Previous studies have shown that brown adipose tissue (BAT) is particularly sensitive to CLA-supplemented diet feeding. Most of them use mixtures containing several CLA isomers, mainly cis-9,trans-11 and trans-10, cis-12 in equal concentration. Our aim was to characterize the separate effects of both CLA isomers on thermogenic capacity in cultured brown adipocytes. The CLA isomers showed opposite effects. Hence, on the one hand,trans-10, cis-12 inhibited uncoupling protein (UCP) 1 induction by norepinephrine (NE) and produced a decrease in leptin mRNA levels. These effects were associated with a blockage of CCAAT-enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ₂ mRNA expression. On the other hand, cis-9, trans-11 enhanced the UCP1 elicited by NE, an effect reported earlier for polyunsaturated fatty acids and also observed here for linoleic acid. These findings could explain, at least in part, the effects observed in vivo when feeding a CLA mixture supplemented diet as a result of the combined action of CLA isomers (reduction of adipogenesis and defective BAT thermogenesis that could be through trans-10, cis-12 and enhanced UCP1 thermogenic capacity through cis-9, trans-11).

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.

Control of blood pressure and of blood flow is essential for maintenance of homeostasis. The hemodynamic state is adjusted by intrinsic, neural, and hormonal mechanisms to optimize adaptation to internal and environmental challenges. In the last decade, many studies showed that modification of the mouse genome may alter the capacity of cardiovascular control systems to respond to homeostatic challenges or even bring about a permanent pathophysiological state. This review discusses the progress that has been made in understanding of autonomic cardiovascular control mechanisms from studies in genetically modified mice. First, from a physiological perspective, we describe how basic hemodynamic function can be measured in conscious conditions in mice. Second, we focus on the integrative role of autonomic nerves in control of blood pressure in the mouse, and finally, we depict the opportunities and insights provided by genetic modification in this area.

The development and widespread use of genetically altered mice to study the role of various proteins in biological control systems have led to a renewed interest in methodologies and approaches for evaluating physiological phenotypes. As a result, cross-disciplinary approaches have become essential for fully realizing the potential of these new and powerful animal models. The combination of classical physiological approaches and modern innovative technology has given rise to an impressive arsenal for evaluating the functional results of genetic manipulation in the mouse. This review attempts to summarize some of the techniques currently being used for measuring cardiovascular, renal, and pulmonary variables in the intact mouse, with specific attention to practical considerations useful for their successful implementation.

In the typical mammal, energy flux, protein metabolism, and renal excretory processes constitute a set of closely linked and quantitatively matched functions. However, this matching has limits, and these limits become apparent when animals adapt to unusual circumstances. The vampire bat and shrew have an extremely high protein intake, and the glomerular filtration rate (GFR) is not commensurate with the large urea load to be excreted. The vampire bat is chronically azotemic (blood urea concentration 27–57 mmol/l); yet there is no information as to how this animal has adjusted to such an azotemic internal environment. A high protein intake should also lead to chronic glomerular hyperfiltration; yet neither animal appears to develop progressive renal failure. The American black bear, on the other hand, has adapted to a prolonged period without intake or urine output. Despite continued amino acid catabolism with urea production, this mammal is able to completely salvage and reutilize urea nitrogen for protein synthesis, although the signals that initiate this metabolic adaptation are not known. The vampire bat, shrew, and bear are natural models adapted to circumstances analogous to chronic renal failure. Unraveling these adaptations could lead to new interventions for the prevention/treatment of chronic renal failure.

Cerebrospinal fluid (CSF) drains through the cribriform plate (CP) in association with the olfactory nerves. From this location, CSF is absorbed into nasal mucosal lymphatics. Recent data suggest that this pathway plays an important role in global CSF transport in sheep. In this report, we tested the hypothesis that blocking CSF transport through this pathway would elevate resting intracranial pressure (ICP). ICP was measured continuously from the cisterna magna of sheep before and after CP obstruction in the same animal. To block CSF transport through the CP, an external ethmoidectomy was performed. The olfactory and adjacent mucosa were removed, and the bone surface was sealed with tissue glue. To restrict our analysis to the cranial CSF system, CSF transport into the spinal subarachnoid compartment was prevented with a ligature tightened around the thecal sac between C1 and C2. Sham surgical procedures had no significant effects, but in the experimental group CP obstruction elevated ICP significantly. Mean postobstruction steady-state pressures (18.0 ± 3.8 cmH₂O) were approximately double the preobstruction values (9.2 ± 0.9 cmH₂O). These data support the concept that the olfactory pathway represents a major site for CSF drainage.

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered α₂-adrenergic receptor (α₂-AR) density and/or affinity. In fetal (∼140 days) and adult sheep, we measured α₂-AR density and affinity with the antagonist [³H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the α₂-AR antagonists yohimbine and idazoxan and contractile responses to the α₂-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, α₂-AR density was 201 ± 18 and 52 ± 6 fmol/mg protein, respectively (P< 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10⁻⁷ M yohimbine significantly decreased the pD₂ for NE-induced tension in the presence of 3 × 10⁻⁵ M cocaine, 10⁻⁵ M deoxycorticosterone, and 10⁻⁶ M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD₂ for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10⁻⁶ M idazoxan to block α₂-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, α₂-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional α₂-AR.

Hemodynamic responses to adenosine, the A₁ receptor agonistsN ⁶-cyclopentyladenosine (CPA) and adenosine amine congener (ADAC), and the A₂ receptor agonist 5′-(N-cyclopropyl)-carboxamido-adenosine (CPCA) were investigated in the hindquarter vascular bed of the cat under constant-flow conditions. Injections of adenosine, CPA, ADAC, CPCA, ATP, and adenosine 5′-O-(3-thiotriphosphate) (ATPγS) into the perfusion circuit induced dose-related decreases in perfusion pressure. Vasodilator responses to the A₁ agonists were reduced by the A₁ receptor antagonists KW-3902 and CGS-15943, whereas responses to CPCA were reduced by the A₂ antagonist KF-17837. Vasodilator responses to adenosine were reduced by KW-3902, CGS-15943, and by KF-17837, suggesting a role for both A₁ and A₂ receptors. Vasodilator responses to ATP and the nonhydrolyzable ATP analog ATPγS were not attenuated by CGS-15943 or KF-17837. After treatment with the nitric oxide synthase inhibitorN ^ω-nitro-l-arginine methyl ester, the cyclooxygenase inhibitor sodium meclofenamate, or the ATP-dependent K⁺ (K ATP + ) channel antagonists U-37883A or glibenclamide, responses to adenosine and ATP were not altered. Responses to adenosine, CPA, and CPCA were increased in duration by rolipram, a type 4 cAMP phosphodiesterase inhibitor, but were not altered by zaprinast, a type 5 cGMP phosphodiesterase inhibitor. When blood flow was interrupted for a 30-s period, the magnitude and duration of the reactive vasodilator response were reduced by A₁ and A₂ receptor antagonists. These data suggest that vasodilator responses to adenosine and the A₁and A₂ agonists studied are not dependent on the release of cyclooxygenase products, nitric oxide, or the opening of K ATP + channels in the regional vascular bed of the cat. The present data suggest a role for cAMP in mediating responses to adenosine and suggest that vasodilator responses to adenosine and to reactive hyperemia are mediated in part by A₁ and A₂ receptors in the hindquarter vascular bed of the cat.

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca²⁺-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excessl-arginine partially recovered both of the impaired relaxations. The contents ofN ^G-monomethyl-l-arginine (l-NMMA) and asymmetricN ^G, N ^G-dimethyl-l-arginine (ADMA) but not l-arginine and symmetricN ^G,N′^G-dimethyl-l-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic l-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess l-arginine abolished the inhibition with l-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of l-NMMA and ADMA.

We studied the effect of troglitazone on cellular acid-base balance and alanine formation in isolated rat mesangial cells. Mesangial cells were grown to confluency in RPMI 1640 media on 30-mm chambers used to monitor both cellular pH using the pH-sensitive dye 2′7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein and metabolic acid production as well as glutamine metabolism. Troglitazone (10 μM) induced a spontaneous cellular acidosis (6.95 ± 0.02 vs. 7.47 ± 0.04, respectively; P < 0.0001) but without an increase in lactic acid production. Alanine production was reduced 64% (P < 0.01) consistent with inhibition of the glutamate transamination. These findings pointed to a decrease in acid extrusion rather than an increase in acid production as the underlying mechanism leading to the cellular acidosis. To test their acid extrusion capabilities, mesangial cells were acid loaded with NH 4 + and then allowed to recover in Krebs-Henseleit media or in Krebs-Henseleit media minus bicarbonate (HEPES substituted), and the recovery response (ΔpH_i/min) was monitored. In the presence of 10 μM troglitazone, the recovery response to the NH 4 + acid load was virtually eliminated in the bicarbonate-buffered media (0.00 ± 0.001 vs. 0.06 ± 0.02 pH_i/min, P < 0.0001 vs. control) and reduced 75% in HEPES-buffered media (0.01 ± 0.01 vs. 0.04 ± 0.02 pH_i/min, P < 0.002 vs. control). These results show that troglitazone induces a spontaneous cellular acidosis resulting from a reduction in cellular acid extrusion.

Agouti-related protein (AgRP) is coexpressed with neuropeptide Y (NPY) in a population of neurons in the arcuate nucleus (ARC) of the hypothalamus and stimulates food intake for up to 7 days if injected intracerebroventricularly. The prolonged food intake stimulation does not seem to depend on continued competition at the melanocortin-4 receptor (MC4R), because the relatively specific MC4R agonist MTII regains its ability to suppress food intake 24 h after AgRP injection. Intracerebroventricular AgRP also stimulates c-Fos expression 24 h after injection in several brain areas, so the neurons exhibiting delayed Fos expression might be particularly important in feeding behavior. Thus we aimed to identify the neurochemical phenotype of some of these neurons in select hypothalamic areas, using double-label immunohistochemistry. AgRP-injected rats ingested significantly more chow (10.2 ± 0.6 g) vs. saline controls (3.4 ± 0.7 g) in the first 9 h (light phase) after injection. In the lateral hypothalamus (particularly the perifornical area) 23 h after injection, AgRP induced significantly more Fos vs. saline in orexin-A (OXA) neurons (25.6 ± 4.9 vs. 4.8 ± 3.1%), but not in melanin-concentrating hormone (MCH) or cocaine- and amphetamine-regulated transcript (CART) neurons. In the ARC, AgRP induced significantly more Fos in CART (40.6 ± 5.9 vs. 13.4 ± 1.8%) but not NPY neurons. In the paraventricular nucleus, there was no significant difference in Fos expression induced by AgRP vs. saline in oxytocin and CART neurons. We conclude that the long-lasting hyperphagia induced by AgRP is correlated with and possibly partially mediated by hyperactive OXA neurons in the lateral hypothalamus and CART neurons in the ARC, but not by NPY and MCH neurons. The substantial increase in light-phase food intake by AgRP supports a role for the arousing effects of OXA. Activation of CART neurons in the ARC (which likely coexpress proopiomelanocortin) could indicate attempts to activate counterregulatory decreases in food intake.

The present experiment investigated Fos-like immunoreactivity (FLI) in the nucleus of the solitary tract (NST) after intraoral infusions of 0.1 M citric acid, 0.3 M NaCl, and 0.3–30 mM quinine monohydrochloride (QHCl) in awake, behaving rats. Increases in QHCl concentration produced increases in the numbers of FLI-labeled neurons in the rostral part of the intermediate (i_r) and rostral (r) NST, but the topographic distribution of FLI was consistent across QHCl concentrations and distinctive compared with effects of citric acid. Quinine elicited FLI concentrated in the medial third of the nucleus; acid elicited more broadly distributed FLI concentrated farther laterally. Surprisingly, in contrast to QHCl and citric acid, NaCl produced FLI that was indistinguishable from that produced by water. Although the functional significance of these patterns is unknown, citric acid and QHCl are nonpreferred stimuli but produced different oromotor behaviors. QHCl (30 mM) elicited ∼3.2 times as many gapes as citric acid (0.1 M), and acid elicited more ingestive responses. Parallel differences in FLI expression suggest that different NST regions may have distinctive roles in triggering oromotor behaviors.

Increases in the concentration of interstitial potassium concentration during exercise may play a role in the modulation of the cardiovascular response to exercise. However, it is not known if changes in potassium correlate with indexes of muscle reflex engagement. Eight healthy subjects performed dynamic [rhythmic handgrip (RHG)] and static handgrip (SHG) exercise at 40% of maximal voluntary contraction. Forearm circulatory arrest was performed to assess the metaboreceptor component of the exercise pressor reflex. Mean arterial pressure (MAP) and muscle sympathetic nerve activity (MSNA) were measured during each exercise paradigm. Venous plasma potassium concentrations ([K⁺]_V) were measured and used as a surrogate marker for interstitial potassium. [K⁺]_V were measured at baseline and at 1-min intervals during dynamic handgrip. During SHG, [K⁺]_V were measured at baseline, 30 and 90 s of exercise, and twice during forearm circulatory arrest. Mean [K⁺]_V was 3.6 mmol/l at rest before both paradigms. During RHG, [K⁺]_V rose by ∼1.0 mmol/l by min 2 and remained constant throughout the rest of handgrip. During SHG, [K⁺]_V rose significantly at 30 s and rose an additional ∼1.0 mmol/l by peak exercise. MAP and MSNA rose during both exercise paradigms. During posthandgrip circulatory arrest (PHG-CA), MSNA and blood pressure remained above baseline. [K⁺]_V and MSNA did not correlate during either exercise paradigm. Moreover, during PHG-CA, there was clear dissociation of MSNA from [K⁺]_V. These data suggest that potassium does not play a direct role in the maintenance of the exercise pressor reflex.

In this study we evaluated by telemetry the effects of ANG II and ANG-(1–7) infusion on the circadian rhythms of blood pressure (BP) and heart rate (HR) and on the cardiovascular adjustment resulting from restraint stress in rats. ANG II or ANG-(1–7) or vehicle were infused subcutaneously for 7 days. Restraint stress was carried out before, during, and after infusion at 7-day intervals. Parallel with an increase in MAP, ANG II infusion produced an inversion of MAP circadian rhythm with a significant MAP acrophase inversion. It also produced bradycardia during the first 3 days of infusion. Thereafter, HR progressively increased, reaching values similar to or above those of the control period at the end of the infusion period. HR circadian variation was not changed by ANG II infusion. Strikingly, ANG II significantly attenuated the increase in MAP induced by restraint stress without altering the HR response. ANG-(1–7) infusion produced a slight but significant decrease in MAP restricted to the daytime period. No significant changes in the MAP acrophase were observed. In addition, ANG-(1–7) infusion produced a small but significant sustained bradycardia. ANG-(1–7) did not change cardiovascular responses to restraint stress. These data indicate that ANG II can influence the activity of brain areas involved in the determination of stress-induced or circadian-dependent variations of blood pressure without changing HR fluctuations. A significant modulatory influence of ANG-(1–7) on basal MAP and HR is also suggested.

The product of heart rate (HR) and systolic blood pressure (SBP), the double product (DP), is an indirect index of cardiac oxygen consumption. We used spectral analysis to test the hypothesis that baroreflex adjustments of HR stabilize the DP during spontaneous variations in SBP. SBP and HR were recorded by telemetry in five male conscious rabbits. HR and SBP power spectra each exhibited a low frequency peak at ∼0.05 Hz that was associated with high (>0.5) spectral coherence and a positive phase relationship between SBP and HR (SBP leading). A prominent peak was absent in the spectra of their product, suggesting that SBP and HR interacted to reduce DP variability in this frequency region. In contrast, a prominent 0.05-Hz peak was present in the power spectrum of calculated surrogates of the DP in which reflex interactions between HR and SBP had been removed. Our results suggest that baroreflex adjustments of HR stabilize the DP during spontaneous low-frequency variations in SBP in conscious rabbits.

Salt restriction leads to parallel increases of renin, cyclooxygenase-2 (COX-2), and neuronal nitric oxide synthase (nNOS) gene expression in the juxtaglomerular apparatus of rat kidneys. Because the upregulation of these genes is strongly enhanced if salt restriction is combined with inhibition of the renin-angiotensin-aldosterone system, our study aimed to find out whether the juxtaglomerular expressions of renin, COX-2, and nNOS are subject to a common direct negative feedback control by ANG II. For this purpose, male Sprague-Dawley rats were fed a low-salt diet (0.02% wt/wt) with or without additional treatment with the ANG I-converting enzyme (ACE) inhibitor ramipril (10 mg · kg body wt⁻¹ · day⁻¹) for 1 wk, and renocortical renin, COX-2, and nNOS mRNAs were assayed. To narrow down possible indirect effects of the ACE inhibitor that may result from insufficient aldosterone production, the animals received mineralocorticoid substitution with fludrocortisone (6 mg · kg body wt⁻¹ · day⁻¹). Thus mineralocorticoid substitution prevented the fall of systolic blood pressure and of glomerular filtration induced by ramipril in rats on low-salt diet. Although fludrocortisone had no effect on basal renin, COX-2, and nNOS mRNA, it clearly attenuated the threefold increases of both renin and COX-2 mRNA in response to low-salt diet. In rats on low-salt diet, ramipril further increased renin mRNA ninefold, COX-2 mRNA fourfold, and nNOS 2.5-fold in the absence of fludrocortisone. In the presence of fludrocortisone, ramipril increased renin mRNA 10-fold, COX-2 mRNA 2.5-fold, and nNOS mRNA 2.5-fold. These data indicate that mineralocorticoid substitution lowers the overall expression of juxtaglomerular renin and COX-2 during low-salt intake and attenuates a further rise of COX-2 expression by ACE inhibition, but it does not change the stimulatory effect of ACE inhibition on renin and nNOS expression. We conclude that the expression of renin, COX-2, and nNOS in the juxtaglomerular apparatus during low-salt diet is markedly limited by a direct feedback inhibition through ANG II.

Increasing renal pelvic pressure increases afferent renal nerve activity (ARNA) by a PGE₂-mediated release of substance P (SP) from renal pelvic nerves. The role of cAMP activation in the PGE₂-mediated release of SP was studied by examining the effects of the adenylyl cyclase (AC) activator forskolin and AC inhibitor dideoxyadenosine (DDA). Forskolin enhanced the bradykinin-mediated release of SP from an isolated rat renal pelvic wall preparation, from 7.3 ± 1.3 to 15.6 ± 3.0 pg/min. PGE₂ at a subthreshold concentration for SP release mimicked the effects of forskolin. The EP₂ receptor agonist butaprost, 15 μM, and PGE₂, 0.14 μM, produced similar increases in SP release, from 5.8 ± 0.8 to 17.0 ± 2.3 pg/min and from 8.0 ± 1.3 to 21.6 ± 2.7 pg/min. DDA blocked the SP release produced by butaprost and PGE₂. The PGE₂-induced release of SP was also blocked by the PKA inhibitors PKI_14–22 and H-89. Studies in anesthetized rats showed that renal pelvic administration of butaprost, 10 μM, and PGE₂, 0.14 μM, resulted in similar ARNA responses, 1,520 ± 390 and 1,170 ± 270% · s (area under the curve of ARNA vs. time) that were blocked by DDA. Likewise, the ARNA response to increased renal pelvic pressure, 7,180 ± 710% · s, was blocked by DDA. In conclusion, PGE₂activates the cAMP-PKA pathway leading to a release of SP and activation of renal pelvic mechanosensory nerve fibers.

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue afterday 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was ∼10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth.

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.

Acclimation of the European lesser-spotted dogfish Scyliorhinus canicula to reduced environmental salinity [85–70% seawater (SW)] induced a significant diuresis in addition to a significant decrease in plasma osmolality in vivo. The threshold for this diuresis was determined to be 85% SW. Therefore,S. canicula acclimated to 85% SW was selected for further study as a diuretic model in the development of an in situ perfused kidney preparation. The renal role of arginine vasotocin (AVT) in the in situ perfused trunk preparation was investigated. In SW, perfusion of 10⁻⁹ and 10⁻¹⁰ M AVT resulted in a glomerular antidiuresis and decreases in tubular transport maxima for glucose and perfusate flow. In 85% SW, 10⁻¹⁰ M AVT had no significant effect on these renal parameters with the exception of transport maxima for glucose and perfusate flow. Tubular parameters remained unchanged by either 10⁻⁹ or 10⁻¹⁰ M AVT. The results demonstrate that the perfused kidney preparation was a viable tool for the investigation of renal parameters in elasmobranch fish and that AVT induced a glomerular antidiuresis.

Juvenile salmon migrating from freshwater to the marine environment confront a marked change in environmental osmolality. Using differential display of mRNA expression, we cloned a 1.9-kb cDNA upregulated in isolated tissues of salmon exposed to the hyperosmotic stress associated with transition to the dehydrating marine environment. The cDNA codes for a 21-kDa protein, salmon hyperosmotic protein 21 (Shop21), with 98% identity to Rbx1, an E3 ubiquitin ligase; the protein also contains a novel 81-amino acid domain at the NH₂ terminus not found in Rbx1. Moderate hyperosmotic stress (24 h at 550 mosmol/kg) increased Shop21 transcript 10-fold in branchial lamellae, whereas no upregulation was observed under more severe stress (≥800 mosmol/kg). Expression of the gene also was observed in heart and kidney. Replacement of NaCl with mannitol, but not glycerol, also elicited an increase in Shop21 mRNA. Inhibition of the mitogen-activated protein kinase and mitogen-activated extracellular regulated kinase kinase signal transduction pathways failed to blunt the Shop21 response during hyperosmotic stress. Shop21 mRNA also accumulated during thermal stress but to a lesser extent than heat shock protein 70 mRNA. The potential importance of Shop21 to the living animal is suggested by marked upregulation of the gene in salmon after transfer to seawater. The results of these investigations suggest that Shop21 may have a role in targeting selected proteins (e.g., in freshwater ionocytes) nonessential for adaptation to seawater for removal via the proteasome pathway.

Cerebral osmoreceptors mediate thirst and neurohypophyseal secretion stimulated by increases in the effective osmolality of plasma (P_osmol). The present experiments determined whether an intragastric load of hypertonic saline (ig HS; 0.5 M NaCl, 4 ml) would potentiate these responses before induced increases in P_osmol in the general circulation could be detected by cerebral osmoreceptors. Adult rats deprived of water overnight and then given intragastric HS consumed much more water in 15–30 min than rats given either pretreatment alone, even though systemic P_osmol had not yet increased significantly because of the gastric load. In other rats pretreated with an intravenous infusion of 1 M NaCl (2 ml/h for 2 h), plasma levels of vasopressin and oxytocin were considerably elevated 15 and 25 min after intragastric HS treatment, whereas systemic P_osmol was not increased further. These and other findings are consistent with previous reports that hepatic portal osmoreceptors (or Na⁺ receptors) stimulate thirst and neurohypophyseal hormone secretion in euhydrated rats given gastric NaCl loads and indicate that these effects are potentiated when animals are dehydrated.

The present study sought to determine whether arterial baroreceptor afferents mediate the inhibitory effect of an acute increase in arterial blood pressure (AP) on thirst stimulated by systemically administered ANG II or by hyperosmolality. Approximately 2 wk after sinoaortic denervation, one of four doses of ANG II (10, 40, 100, or 250 ng · kg⁻¹ · min⁻¹) was infused intravenously in control and complete sinoaortic-denervated (SAD) rats. Complete SAD rats ingested more water than control rats when infused with 40, 100, or 250 ng · kg⁻¹ · min⁻¹ ANG II. Furthermore, complete SAD rats displayed significantly shorter latencies to drink compared with control rats. In a separate group of rats, drinking behavior was stimulated by increases in plasma osmolality, and mean AP was raised by an infusion of phenylephrine (PE). The infusion of PE significantly reduced water intake and lengthened the latencies to drink in control rats but not in complete SAD rats. In all experiments, drinking behavior of rats that were subjected to sinoaortic denervation surgery but had residual baroreceptor reflex function (partial SAD rats) was similar to that of control rats. Thus it appears that arterial baroreceptor afferents mediate the inhibitory effect of an acute increase in AP on thirst stimulated by ANG II or hyperosmolality.

The adrenomedullin (AM) preprohormone is posttranslationally processed to result in two biologically active fragments, AM and proadrenomedullin NH₂-terminal 20 peptide (PAMP). AM is thought to play a role in fluid and electrolyte balance by acting in brain to inhibit salt and water appetite and in the kidney to cause diuresis and natriuresis. We previously have shown that AM is necessary for the short-term regulation of salt intake. In this paper, we have designed a ribozyme, a catalytic RNA molecule, which specifically recognizes and cleaves the AM transcript. In cultured vascular smooth muscle cells, ribozyme treatment lowered AM mRNA and reduced peptide content. Intracerebroventricular administration of the ribozyme lowered hypothalamic AM content and led to an exaggerated drinking response in rats, demonstrating that endogenous, brain-derived AM is physiologically relevant and necessary for short-term control of water intake.

The hypothesis that increases in plasma sodium induce natriuresis independently of changes in body fluid volume was tested in six slightly dehydrated seated subjects on controlled sodium intake (150 mmol/day). NaCl (3.85 mmol/kg) was infused intravenously over 90 min as isotonic (Iso) or as hypertonic saline (Hyper, 855 mmol/l). After Hyper, plasma sodium increased by 3% (142.0 ± 0.6 to 146.2 ± 0.5 mmol/l). During Iso a small decrease occurred (142.3 ± 0.6 to 140.3 ± 0.7 mmol/l). Iso increased estimates of plasma volume significantly more than Hyper. However, renal sodium excretion increased significantly more with Hyper (291 ± 25 vs. 199 ± 24 μmol/min). This excess was not mediated by arterial pressure, which actually decreased slightly. Creatinine clearance did not change measurably. Plasma renin activity, ANG II, and aldosterone decreased very similarly in Iso and Hyper. Plasma atrial natriuretic peptide remained unchanged, whereas plasma vasopressin increased with Hyper (1.4 ± 0.4 to 3.1 ± 0.5 pg/ml) and decreased (1.3 ± 0.4 to 0.6 ± 0.1 pg/ml) after Iso. In conclusion, the natriuretic response to Hyper was 50% larger than to Iso, indicating that renal sodium excretion may be determined partly by plasma sodium concentration. The mechanism is uncertain but appears independent of changes in blood pressure, glomerular filtration rate, the renin system, and atrial natriuretic peptide.