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To test the hypothesis that increased osmolality contributes to hypertension in deoxycorticosterone acetate (DOCA)-salt-hypertensive rats by acting in the brain, DOCA-salt and Sham-salt rats were instrumented with bilateral, nonoccluding intracarotid and femoral catheters. Two weeks prior, rats were uninephrectomized and received subcutaneous implants with or without DOCA (65 mg) and began drinking salt water (1% NaCl and 0.2% KCl). DOCA-salt rats (n=28) exhibited elevated blood pressure (159+/-4 mm Hg; P<0.05) and heart rate (392+/-10 bpm; P<0.05) compared with Sham-salt animals (n=5; blood pressure: 107+/-5 mm Hg; heart rate: 355+/-10 bpm). Bilateral intracarotid infusion of hypotonic fluid (osmolality: approximately 40 mOsm/L), which lowers osmolality of blood to the brain by approximately 2%, rapidly decreased blood pressure in DOCA-salt rats (-22+/-4 mm Hg after 15 minutes; P<0.05; n=7) but not Sham-salt rats (2+/-2 mm Hg; n=5). Hypotonic fluid infused intravenously did not lower blood pressure (0+/-2 mm Hg) in DOCA-salt rats (n=7). In DOCA-salt rats pretreated with a V(1) vasopressin antagonist (Manning compound, 5 microg, IV), intracarotid hypotonic infusion still decreased blood pressure (-10+/-3 mm Hg; P<0.05; n=9), but the response was smaller (P<0.05). Finally, in DOCA-salt rats (n=4) pretreated with the V(1) antagonist and the ganglionic blocker hexamethonium, decreasing osmolality of blood to the brain did not reduce blood pressure. These data indicate that, in DOCA-salt rats, hypertonicity acts in the brain to support blood pressure, in part by stimulating vasopressin secretion and in part by stimulating another rapidly reversible mechanism, likely the sympathetic nervous system.

Using deoxycorticosterone acetate (DOCA)-salt rats, we tested the hypothesis that increased plasma NaCl concentration supports sympathetic activity and blood pressure (BP) during salt-sensitive hypertension. One day before experimentation, femoral catheters and an electrode for measurement of lumbar sympathetic nerve activity (LSNA) probe were surgically positioned in DOCA-salt and Sham-salt rats. DOCA-salt rats exhibited increased (P<0.05) BP and NaCl concentration (BP, 163+/-8 mm Hg; NaCl, 260.8+/-3.3 mEq/L [DOCA-salt]: BP, 106.3+/-4.2 mm Hg; NaCl, 254.3+/-1.7 mEq/L [Sham-salt]). After V1 vasopressin blockade (Manning compound, 5 microg IV), infusion (0.12 mL/min) of 5% dextrose in water decreased NaCl concentrations, BP (-28+/-7 mm Hg), and LSNA (-39+/-5%) in DOCA-salt but not Sham-salt rats. To explain how such small (approximately 2%) increases in plasma NaCl could underlie the hypertension, we hypothesized that DOCA augments the pressor and sympathoexcitatory actions of NaCl. To address this hypothesis, animals with equally elevated NaCl but no DOCA (Sham-1.7% salt) and animals with increased DOCA but normal NaCl levels (DOCA-water) were prepared and administered the infusion of 5% dextrose in water. BP and LSNA were not altered in DOCA-water rats. In the Sham-1.7% salt rats, BP fell (P<0.05), but not LSNA, and the responses were significantly smaller than that observed in the DOCA-salt animals. Collectively, these data suggest that increased NaCl levels contribute to sympathoexcitation and hypertension in DOCA-salt rats because of amplification of the NaCl signal by DOCA.

To test the hypothesis that high osmolality acts in the brain to chronically support mean arterial pressure (MAP) and lumbar sympathetic nerve activity (LSNA), the osmolality of blood perfusing the brain was reduced in conscious water-deprived and water-replete rats by infusion of hypotonic fluid via bilateral nonoccluding intracarotid catheters. In water-deprived rats, the intracarotid hypotonic infusion, estimated to lower osmolality by approximately 2%, decreased MAP by 9+/-1 mmHg and LSNA to 86+/-7% of control; heart increased by 25+/-8 beats per minute (bpm) (all P<0.05). MAP, LSNA, and heart rate did not change when the hypotonic fluid was infused intravenously. The intracarotid hypotonic fluid infusion was also ineffective in water-replete rats. Prior treatment with a V1 vasopressin antagonist did not alter the subsequent hypotensive and tachycardic effects of intracarotid hypotonic fluid infusion in water-deprived rats. In summary, acute decreases in osmolality of the carotid blood of water-deprived, but not water-replete, rats decreases MAP and LSNA and increases heart rate. These data support the hypothesis that the elevated osmolality induced by water deprivation acts via a region perfused by the carotid arteries, presumably the brain, to tonically increase MAP and LSNA and suppress heart rate.

Water deprivation is associated with increased excitatory amino acid (EAA) drive of the rostral ventrolateral medulla (RVLM), but the mechanism is unknown. This study tested the hypotheses that the increased EAA activity is mediated by decreased blood volume and/or increased osmolality. This was first tested in urethane-anesthetized rats by determining whether bilateral microinjection of kynurenate (KYN, 2.7 nmol) into the RVLM decreases arterial pressure less in water-deprived rats after normalization of blood volume by intravenous infusion of isotonic saline or after normalization of plasma osmolality by intravenous infusion of 5% dextrose in water (5DW). Water-deprived rats exhibited decreased plasma volume and elevated plasma osmolality, hematocrit, and plasma sodium, chloride, and protein levels (all P < 0.05). KYN microinjection decreased arterial pressure by 24 +/- 2 mmHg (P < 0.05; n = 17). The depressor response was not altered following isotonic saline infusion but, while still present (P < 0.05), was reduced (P < 0.05) to -13 +/- 2 mmHg soon after 5DW infusion. These data suggest that the high osmolality, but not low blood volume, contributes to the KYN depressor response. To further investigate the action of increased osmolality on EAA input to RVLM, water-replete rats were also studied after hypertonic saline infusion. Whereas KYN microinjection did not decrease pressure immediately following the infusion, a depressor response gradually developed over the next 3 h. Lumbar sympathetic nerve activity also gradually increased to up to 167 +/- 19% of control (P < 0.05) 3 h after hypertonic saline infusion. In conclusion, acute and chronic increases in osmolality appear to increase EAA drive of the RVLM.

Nitric oxide (NO) appears to inhibit sympathetic tone in anesthetized rats. However, whether NO tonically inhibits sympathetic outflow, or whether endogenous angiotensin II (ANG II) promotes NO-mediated sympathoinhibition in conscious rats is unknown. To address these questions, we determined the effects of NO synthase (NOS) inhibition on renal sympathetic nerve activity (RSNA) and heart rate (HR) in conscious, unrestrained rats on normal (NS), high-(HS), and low-sodium (LS) diets, in the presence and absence of an ANG II receptor antagonist (AIIRA). When arterial pressure was kept at baseline with intravenous hydralazine, NOS inhibition with l-NAME (10 mg/kg i.v.) resulted in a profound decline in RSNA, to 42 +/- 11% of control (P < 0.01), in NS animals. This effect was not sustained, and RSNA returned to control levels by 45 min postinfusion. l-NAME also caused bradycardia, from 432 +/- 23 to 372 +/- 11 beats/min postinfusion (P < 0.01), an effect, which, in contrast, was sustained 60 min postdrug. The effects of NOS inhibition on RSNA and HR did not differ between NS, HS, and LS rats. However, when LS and HS rats were pretreated with AIIRA, the initial decrease in RSNA after l-NAME infusion was absent in the LS rats, while the response in the HS group was unchanged by AIIRA. These findings indicate that, in contrast to our hypotheses, NOS activity provides a stimulatory input to RSNA in conscious rats, and that in LS animals, but not HS animals, this sympathoexcitatory effect of NO is dependent on the action of endogenous ANG II.

Chronic hypoxia (CH) is associated with both blunted agonist-induced and myogenic vascular reactivity, possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the cellular location of the HO responsible for these effects has not been clearly established. Therefore, we examined the response to administration of the substrate for HO, heme-l-lysinate (HLL), in endothelium-intact and endothelium-denuded small mesenteric arteries from CH male Sprague-Dawley rats. Mesenteric arteries were isolated and mounted on glass cannulas, pressurized to 60 mmHg, and superfused with physiological saline solution. All experiments were performed in the presence of 100 microM N(omega)-nitro-l-arginine. The vasodilator response to HLL or exogenous CO was examined. HLL experiments were performed in the presence and absence of the HO inhibitor zinc protoporphyrin IX (ZnPPIX). HLL administration resulted in a dose-dependent vasodilator response that was abolished in the presence of ZnPPIX or by endothelial removal. Exogenous CO produced a vasodilator response that was independent of an intact endothelium. Cellular localization of HO was verified through immunohistochemistry in sections of the gut and aorta from CH and control animals. Staining for HO-1, HO-2, and endothelial nitric oxide synthase was confined to the endothelium. Thus we conclude that CO is a product of HO located within the endothelium.

We have developed a laboratory exercise that demonstrates arterial baroreflex control of heart rate (HR) in the conscious unrestrained rat, incorporating graduate level physiological topics as well as a hands-on exposure to conscious animal research. This demonstration utilizes rats chronically instrumented to measure cardiac output (CO), HR, and arterial blood pressure in response to agents that raise or lower blood pressure. The HR response to progressive increases or decreases in blood pressure is recorded, and a baroreflex curve is generated by plotting mean arterial blood pressure (MABP) vs. HR. Observation of altered CO allows for discussion of the relationship between MAP, CO, HR, stroke volume, and total peripheral resistance. Administration of arginine vasopressin demonstrates the ability of this hormone to alter the sensitivity of the baroreflex. Throughout the demonstration, students answer questions from a handout about general cardiovascular physiology, specific pathways of agonists, and the baroreflex system, encouraging group and individual critical analysis of the results. Interpretation of the data reemphasizes lecture material and allows students to observe the baroreflex response in a physiological setting.

Rats exposed to chronic hypoxia (CH; 4 wk at 0.5 atm) exhibit attenuated renal vasoconstrictor reactivity to phenylephrine (PE). Preliminary studies from our laboratory suggest that this response is mediated by hypoxic induction of heme oxygenase (HO) and subsequent release of the endogenous vasodilator carbon monoxide. Because vascular HO mRNA is increased within hours of hypoxic exposure, we hypothesized that the onset of reduced reactivity may occur fairly rapidly and correlate with HO expression. Therefore, we examined the onset of attenuated vasoconstriction on CH exposure as well as the duration of hyporeactivity on return to a normoxic environment. Renal vascular resistance (RVR) responses to graded intravenous infusion of PE were measured in conscious rats under control conditions and after 24 h, 48 h, and 4 wk of CH exposure. Vasoreactivity responses were also determined in 4-wk CH rats 1, 5, 24, and 96 h after return to normoxia. We found that RVR responses to PE were significantly blunted after 48 h and 4 wk but not after 24 h of hypoxic exposure. Inhibition of HO with zinc protoporphyrin IX increased RVR and decreased renal blood flow in 48-h CH rats but not controls. Although reactivity to PE was gradually restored after 4 wk of CH, responsiveness was still slightly blunted at 96 h after return to normoxia. Western blot analysis demonstrated a correlation between HO-1 protein levels and attenuated vasoconstrictor response in CH and posthypoxic rats. These data suggest that the onset and offset of physiologically relevant vascular HO expression occur within 2--3 days.

Chronic hypoxia (CH) attenuates systemic vasoconstriction to a variety of agonists in conscious rats. Recent evidence suggests that similarly diminished responses to vasoconstrictors in aortic rings from CH rats may be due to increased endothelial heme oxygenase (HO) activity and enhanced production of the vasodilator carbon monoxide (CO). Thus we hypothesized that a hypoxia-induced increase in HO activity is responsible for decreased vasoconstrictor responsiveness observed in conscious CH rats. CH (4 wk at 0.5 atm) and control rats were renal denervated and instrumented for the measurement of renal blood flow (RBF) and blood pressure. First, renal vasoconstrictor responses to graded intravenous infusion of phenylephrine (PE) were assessed in conscious rats. CH rats demonstrated significantly diminished renal vasoconstrictor responses to PE compared with control responses that persisted even with acute restoration of normoxia. In additional experiments, CH rats exhibited increased renal vascular resistance and decreased RBF in response to the HO inhibitor zinc protoporphyrin IX (11 micromol/kg iv), whereas renal hemodynamics were unaffected by the inhibitor in control animals. Furthermore, we demonstrated greater HO enzyme activity in renal tissue from CH rats compared with controls. These data suggest that enhanced HO activity contributes a tonic vasodilatory influence in the renal vasculature of CH rats that may be responsible for the diminished sensitivity to vasoconstrictor agonists observed under these conditions.

Previous studies suggest that inducible (i) nitric oxide synthase (NOS) expression within the pulmonary vasculature is increased in rats with chronic hypoxia (CH)-induced pulmonary hypertension. We therefore hypothesized that enhanced iNOS expression associated with CH causes attenuated pulmonary vasoconstrictor responsiveness. To test this hypothesis, we examined the effect of selective iNOS blockade with L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) and nonselective NOS inhibition with Nomega-nitro-L-arginine (L-NNA) on vasoconstrictor responses to U-46619 in isolated saline-perfused lungs from both control and CH (4 wk at 380 mmHg) rats. We additionally measured pulmonary hemodynamic responses to L-NIL in conscious CH rats (fraction of inspired O2 = 0.12). Finally, iNOS mRNA levels were assessed in lungs from each group of rats using ribonuclease protection assays. Despite a significant increase in iNOS mRNA expression after exposure to CH, responses to U-46619 were unaltered by L-NIL but augmented by L-NNA in lungs from both control and CH rats. Pulmonary hemodynamics were similarly unaltered by L-NIL in conscious CH rats. We conclude that iNOS does not modulate pulmonary vasoconstrictor responsiveness after long-term hypoxic exposure.