DNA damage and aging on hematopoietic stem cells: Impact of oxidative stress in ApoE-/- mice.

The effects of aging on ROS production and DNA damage were assessed in hematopoietic stem cells (HSCs) from apolipoprotein E-deficient (ApoE-/-) mice (2-, 12- and 24-month-old), a traditional experimental model of atherogenic dyslipidemia. HSCs from aged ApoE-/- mice were associated with increased ROS levels, leading to loss quiescence, DNA damage, apoptosis and telomere shortening. The concurrence of lack of ApoE and aging result in exhaustion and senescence of HSCs accompanied by increased oxidative stress and inflammation. Therefore, our data open avenues to a better understanding of age-related changes and genetic factors, which may synergistically compromise the efficacy of aged HSC recovery and/or transplantation.

Role of pulmonary stretch receptors and sympathetic system in the inhibition of reflex bradycardia produced by chemical stimulation of the periaqueductal gray matter of the rat.

The present study examined the role of the sympathetic system and pulmonary afferent feedback in the baroreflex inhibition by chemical stimulation of the dorsal periaqueductal gray matter (DPAG) of the anesthetized rat. The baroreflex bradycardia was induced by phenylephrine infusions (PHE, 50 µg/ml/min, i.v.) given either alone or combined with glutamate microinjections (GLU, 10 nmol/100 nl) into the DPAG. GLU microinjections alone produced marked increases in respiratory amplitude (67±19%), but barely changed the respiratory frequency (15±3 cpm) and blood pressure (14±2 mm Hg), and did not affect the heart rate. In contrast, the same injections produced a 92% inhibition of PHE-induced bradycardia (from -62 to -5 bpm). Because GLU microinjections per se had little effects on blood pressure, the baroreflex inhibition should be credited to the deactivation of both the vagal and sympathetic reflex pathways at the medulla. Indeed, the baroreflex was inhibited in only 47% following the DPAG stimulation of atenolol-treated rats. The GLU-evoked inhibition of baroreflex was also correlated with concomitant increases in respiratory amplitude. The role of pulmonary feedback in baroreflex inhibition was thus examined before and after the neuromuscular blockade of atenolol-treated rats. In spontaneously breathing rats, GLU microinjections reversed PHE-induced bradycardia to tachycardia, thereby producing a 153% inhibition of reflex bradycardia (from -38 bpm to +20 bpm). In contrast, the baroreflex inhibition was attenuated in only 53% after neuromuscular blockade (from -34 to -16 bpm). Data are the first evidence of the contribution of pulmonary stretch receptor feedback in DPAG-evoked inhibition of reflex bradycardia.

Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice

The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune®, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg·kg-1·day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 ± 4 vs 124 ± 10 mmHg, respectively) or ACh- (maximal response: 51 ± 8 vs 53 ± 5 percent, respectively) and SNP-induced vasorelaxation (maximal response: 73 ± 6 vs 74 ± 6 percent, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 ± 59 vs 722 ± 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 ± 12 vs 68 ± 8 µm² x 10³). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.

Oral rapamycin attenuates atherosclerosis without affecting the arterial responsiveness of resistance vessels in apolipoprotein E-deficient mice.

The objective of the present study was to assess the effects of the immunosuppressant rapamycin (Rapamune, Sirolimus) on both resistance vessel responsiveness and atherosclerosis in apolipoprotein E-deficient 8-week-old male mice fed a normal rodent diet. Norepinephrine (NE)-induced vasoconstriction, acetylcholine (ACh)- and sodium nitroprusside (SNP)-induced vasorelaxation of isolated mesenteric bed, and atherosclerotic lesions were evaluated. After 12 weeks of orally administered rapamycin (5 mg.kg-1.day-1, N = 9) and compared with untreated (control, N = 9) animals, rapamycin treatment did not modify either NE-induced vasoconstriction (maximal response: 114 +/- 4 vs 124 +/- 10 mmHg, respectively) or ACh- (maximal response: 51 +/- 8 vs 53 +/- 5%, respectively) and SNP-induced vasorelaxation (maximal response: 73 +/- 6 vs 74 +/- 6%, respectively) of the isolated vascular mesenteric bed. Despite increased total cholesterol in treated mice (982 +/- 59 vs 722 +/- 49 mg/dL, P < 0.01), lipid deposition on the aorta wall vessel was significantly less in rapamycin-treated animals (37 +/- 12 vs 68 +/- 8 microm(2) x 10(3)). These results indicate that orally administered rapamycin is effective in attenuating the progression of atherosclerotic plaque without affecting the responsiveness of resistance vessels, supporting the idea that this immunosuppressant agent might be of potential benefit against atherosclerosis in patients undergoing therapy.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension.

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan [10 mg x kg(-1) x day(-1); N = 5] or the superoxide scavenger tempol [0.2 mmol x kg(-1) x day(-1); N = 5], which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats.

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 +/- 4 vs D: 115 +/- 4 mmHg) or HR (S: 340 +/- 12 vs D: 368 +/- 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 +/- 1 mmHg and -16 +/- 2 bpm vs D: -16 +/- 1 mmHg and 9 +/- 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 +/- 9%) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 ± 4 vs D: 115 ± 4 mmHg) or HR (S: 340 ± 12 vs D: 368 ± 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 ± 1 mmHg and -16 ± 2 bpm vs D: -16 ± 1 mmHg and 9 ± 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 ± 9 percent) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Transitory increased blood pressure after upper airway surgery for snoring and sleep apnea correlates with the apnea-hypopnea respiratory disturbance index.

A transitory increase in blood pressure (BP) is observed following upper airway surgery for obstructive sleep apnea syndrome but the mechanisms implicated are not yet well understood. The objective of the present study was to evaluate changes in BP and heart rate (HR) and putative factors after uvulopalatopharyngoplasty and septoplasty in normotensive snorers. Patients (N = 10) were instrumented for 24-h ambulatory BP monitoring, nocturnal respiratory monitoring and urinary catecholamine level evaluation one day before surgery and on the day of surgery. The influence of postsurgery pain was prevented by analgesic therapy as confirmed using a visual analog scale of pain. Compared with preoperative values, there was a significant (P < 0.05) increase in nighttime but not daytime systolic BP (119 5 vs 107 3 mmHg), diastolic BP (72 4 vs 67 2 mmHg), HR (67 4 vs 57 2 bpm), respiratory disturbance index (RDI) characterized by apnea-hypopnea (30 10 vs 13 4 events/h of sleep) and norepinephrine levels (22.0 4.7 vs 11.0 1.3 g l-1 12 h-1) after surgery. A positive correlation was found between individual variations of BP and individual variations of RDI (r = 0.81, P < 0.01) but not between BP or RDI and catecholamines. The visual analog scale of pain showed similar stress levels on the day before and after surgery (6.0 0.8 vs 5.0 0.9 cm, respectively). These data strongly suggest that the cardiovascular changes observed in patients who underwent uvulopalatopharyngoplasty and septoplasty were due to the increased postoperative RDI.

Transitory increased blood pressure after upper airway surgery for snoring and sleep apnea correlates with the apnea-hypopnea respiratory disturbance index

A transitory increase in blood pressure (BP) is observed following upper airway surgery for obstructive sleep apnea syndrome but the mechanisms implicated are not yet well understood. The objective of the present study was to evaluate changes in BP and heart rate (HR) and putative factors after uvulopalatopharyngoplasty and septoplasty in normotensive snorers. Patients (N = 10) were instrumented for 24-h ambulatory BP monitoring, nocturnal respiratory monitoring and urinary catecholamine level evaluation one day before surgery and on the day of surgery. The influence of postsurgery pain was prevented by analgesic therapy as confirmed using a visual analog scale of pain. Compared with preoperative values, there was a significant (P < 0.05) increase in nighttime but not daytime systolic BP (119 ± 5 vs 107 ± 3 mmHg), diastolic BP (72 ± 4 vs 67 ± 2 mmHg), HR (67 ± 4 vs 57 ± 2 bpm), respiratory disturbance index (RDI) characterized by apnea-hypopnea (30 ± 10 vs 13 ± 4 events/h of sleep) and norepinephrine levels (22.0 ± 4.7 vs 11.0 ± 1.3 æg l-1 12 h-1) after surgery. A positive correlation was found between individual variations of BP and individual variations of RDI (r = 0.81, P < 0.01) but not between BP or RDI and catecholamines. The visual analog scale of pain showed similar stress levels on the day before and after surgery (6.0 ± 0.8 vs 5.0 ± 0.9 cm, respectively). These data strongly suggest that the cardiovascular changes observed in patients who underwent uvulopalatopharyngoplasty and septoplasty were due to the increased postoperative RDI.

Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise.

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension.

Ambulatory blood pressure and Doppler echocardiographic indexes of borderline hypertensive men presenting an exaggerated blood pressure response during dynamic exercise

Borderline hypertension (BH) has been associated with an exaggerated blood pressure (BP) response during laboratory stressors. However, the incidence of target organ damage in this condition and its relation to BP hyperreactivity is an unsettled issue. Thus, we assessed the Doppler echocardiographic profile of a group of BH men (N = 36) according to office BP measurements with exaggerated BP in the cycloergometric test. A group of normotensive men (NT, N = 36) with a normal BP response during the cycloergometric test was used as control. To assess vascular function and reactivity, all subjects were submitted to the cold pressor test. Before Doppler echocardiography, the BP profile of all subjects was evaluated by 24-h ambulatory BP monitoring. All subjects from the NT group presented normal monitored levels of BP. In contrast, 19 subjects from the original BH group presented normal monitored BP levels and 17 presented elevated monitored BP levels. In the NT group all Doppler echocardiographic indexes were normal. All subjects from the original BH group presented normal left ventricular mass and geometrical pattern. However, in the subjects with elevated monitored BP levels, fractional shortening was greater, isovolumetric relaxation time longer, and early to late flow velocity ratio was reduced in relation to subjects from the original BH group with normal monitored BP levels (P<0.05). These subjects also presented an exaggerated BP response during the cold pressor test. These results support the notion of an integrated pattern of cardiac and vascular adaptation during the development of hypertension

Cardiovascular neural reflexes in L-NAME-induced hypertension in mice.

The mouse is the most used animal for studying the genetic basis of cardiovascular diseases. However, the mechanisms of regulation of cardiovascular function in this animal are not yet well understood. The goal of this study was to evaluate the baroreflex, the Bezold-Jarisch cardiopulmonary reflex (BJR), and the chemoreflex in mice with hypertension induced by inhibition of NO using Nomega-nitro-L-arginine-methyl ester (L-NAME). Basal mean arterial pressure (MAP) measured under anesthesia (urethane, 1 mg/g IP) was significantly higher in L-NAME (400 microgram/g IP for 7 days)-treated (HT) mice (n=7) compared with vehicle-treated (NT; n=10) animals (126+/-9 versus 79+/-2 mm Hg) without differences in heart rate (HR). Baroreflex sensitivity, evaluated using phenylephrine (1 microgram/g IV) was enhanced in HT mice compared with NT mice (-9.8+/-1.4 versus -4.9+/-0.5 bpm/mm Hg). The BJR, induced by phenylbiguanide (40 ng/g IV), was significantly attenuated in HT animals (MAP, -13+/-5%; HR, -39+/-6%) compared with NT animals (MAP, -38+/-5%; HR, -66+/-2%). The chemoreflex, induced by potassium cyanide (0.26 microgram/g IV), was significantly attenuated in HT animals (MAP, +14+/-4%; HR, -8+/-2%) compared with NT animals (MAP, +29+/-4%; HR, -15+/-4%). As has been observed in rats, chronic inhibition of NO synthase in mice results in arterial hypertension. Enhancement of baroreflex sensitivity and attenuation of BJR and chemoreflex seem to be mainly caused by inhibition of NO synthesis because individual analyses did not show positive correlation between changes in these reflexes and MAP levels in the HT group.

Adenovirus-mediated gene delivery to cells of the magnocellular hypothalamo-neurohypophyseal system.

The objective of the present study was to define the optimum conditions for using replication-defective adenovirus (Ad) to transfer the gene for the green fluorescent protein (GFP) to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and cells of the neurohypophysis (NH). As indicated by characterizing cell survival over 15 days in culture and in electrophysiological whole cell patch-clamp studies, viral concentrations up to 2 x 10(7) pfu/coverslip did not affect viability of transfected PVN and NH cultured cells from preweanling rats. At 2 x 10(7) pfu, GFP gene expression was higher (40% of GFP-positive cells) and more sustained (up to 15 days). Using a stereotaxic approach in adult rats, we were able to directly transduce the PVN, SON, and NH and visualize gene expression in coronal brain slices and in the pituitary 4 days after injection of Ad. In animals receiving NH injections of Ad, the virus was retrogradely transported to PVN and SON neurons as indicated by the appearance of GFP-positive neurons in cultures of dissociated cells from those brain nuclei and by polymerase chain reaction and Western blot analyses of PVN and SON tissues. Adenoviral concentrations of up to 8 x 10(6) pfu injected into the NH did not affect cell viability and did not cause inflammatory responses. Adenoviral injection into the pituitary enabled the selective delivery of genes to the soma of magnocellular neurons. The experimental approaches described here provide potentially useful strategies for the treatment of disordered expression of the hormones vasopressin or oxytocin.

The diuretic chlorthalidone normalizes baroreceptor and Bezold-Jarisch reflexes in DOCA-salt hypertensive rats.

The contributions of arterial baroreceptor and Bezold-Jarisch reflexes, and atrial natriuretic factor (ANF) to the anti-hypertensive effect of the diuretic chlorthalidone were investigated in rats with deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Chlorthalidone (8 mg rat(-1)day(-1)added to food) was given to one group during all 20 days of DOCA (8 mg kg(-1)s.c. twice per week) administration (preventive regimen) and, to another group, 20 days after DOCA treatment was initiated until the 40th day (therapeutic regimen). DOCA caused a significant increase in mean arterial pressure, reduced arterial baroreflex, and increased both the Bezold-Jarisch reflex and pro-ANF converting enzyme activity. Chlorthalidone reversed or prevented the DOCA-salt-induced hypertension, which was accompanied by the normalization of both the arterial baroreflex and the Bezold-Jarisch reflex. Additionally, both preventive and therapeutic regimens with chlorthalidone did cause normalization of the plasma sodium concentration and pro-ANF converting enzyme activity in the left atrium that follows DOCA-salt hypertension. Although it is difficult to determine the relative importance of each of the above regulatory mechanisms altered by chlorthalidone treatment, these data indicate that they may account for the prevention or decrease of DOCA-salt-induced hypertension in rats.

Heated humidification or face mask to prevent upper airway dryness during continuous positive airway pressure therapy.

STUDY OBJECTIVES: The objectives of this study were (1) to evaluate the way in which nasal continuous positive airway pressure (CPAP) therapy influences the relative humidity (rH) of inspired air; and (2) to assess the impact on rH of the addition of an integrated heated humidifier or a full face mask to the CPAP circuitry. DESIGN: The studies were performed in 25 patients with obstructive sleep apnea syndrome receiving long-term nasal CPAP therapy and complaining of nasal discomfort. During CPAP administration, temperature and rH were measured in the mask either during a night's sleep for 8 patients or during a daytime study in which the effects of mouth leaks were simulated in 17 patients fitted with either a nasal mask (with or without humidification) or a face mask alone. SETTING: University hospital sleep disorders center. MEASUREMENTS AND RESULTS: Compared with the values obtained with CPAP alone, integrated heated humidification significantly increased rH during the sleep recording, both when the mouth was closed (60 +/- 14% to 81 +/- 14%, p < 0.01) and during mouth leaks (43 +/- 12% to 64 +/- 8%, p < 0.01). During the daytime study, a significant decrease in rH was observed with CPAP alone. Compared with the values measured during spontaneous breathing without CPAP (80 +/- 2%), the mean rH was 63 +/- 9% (p < 0.01) with the mouth closed and 39 +/- 9% (p < 0. 01) with the mouth open. The addition of heated humidification to CPAP prevented rH changes when the mouth was closed (82 +/- 12%), but did not fully prevent the rH decrease during simulation of mouth leaks (63 +/- 9%) compared with the control period (80 +/- 2%, p < 0. 01). Finally, attachment of a face mask to the CPAP circuitry prevented rH changes both with the mouth closed (82 +/- 9%) and with the mouth open (84 +/- 8%). CONCLUSIONS: These data indicate that inhaled air dryness during CPAP therapy can be significantly attenuated by heated humidification, even during mouth leaks, and can be totally prevented by using a face mask.

L-arginine restores the effect of ouabain on baroreceptor activity and prevents hypertension.

In spontaneously hypertensive rats, ouabain exerts an excitatory effect on baroreceptor nerve activity (BNA). The aim of this study was to determine the effects of ouabain on BNA in other experimental models of hypertension and its interaction with nitric oxide. Rats were made hypertensive using the procedures for N(omega)-nitro-L-arginine methyl ester (L-NAME), deoxycorticosterone acetate (DOCA) salt, and 2-kidney, 1 clip (2K1C) hypertension models. In these groups, systolic arterial pressure was 195+/-7, 149+/-6, and 148+/-4 mm Hg, respectively, compared with 110+/-4 mm Hg in normotensive rats. Acute ouabain administration had an excitatory effect on BNA in normotensive rats (37+/-4%), an inhibitory effect in L-NAME hypertensive rats (-60+/-7%), and no effect in DOCA-salt and 2K1C hypertensive rats. The effects of ouabain were not related to arterial pressure levels, and no excitatory effect on BNA was observed in prehypertensive DOCA-salt rats. Long-term administration of L-arginine (3 g x kg(-1) x day(-1)) prevented DOCA-salt (121+/-8 mm Hg) and 2K1C (104+/-4 mm Hg) hypertension, markedly attenuated L-NAME (130+/-9 mm Hg) hypertension, and restored the excitatory effect of ouabain on BNA in these groups to levels similar to the normotensive rats and their respective control groups. We conclude that ouabain has a diverse effect on BNA in experimental models of hypertension, and it can be normalized by L-arginine. The data also indicate that nitric oxide may play a pivotal role in mediating the excitatory effect of ouabain on BNA, and we speculate that a therapeutic combination of ouabain and L-arginine may be beneficial in secondary hypertension.

Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice.

Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

Cardiovascular changes following acute and chronic chemical lesions of the dorsal periaqueductal gray in conscious rats.

This study was carried out to investigate the effects of chemical lesions of dorsal periaqueductal gray (DPAG) on resting arterial pressure (AP) and heart rate (HR) as well as on cardiac baroreflex of conscious normotensive rats. Lesions were performed by bilateral microinjections of 150 mM NMDA into the DPAG (DPAG-lesion group). Controls were similarly injected with 165 mM NaCl (DPAG-sham group). Animals with chronic lesions confined only to the superior colliculus (SC-lesion group) were also used as controls of DPAG-lesion. Cardiovascular parameters were recorded 1 or 7 days after the microinjections of NMDA in acute and chronic groups, respectively. Cardiac baroreflex was assessed by measuring the HR responses to the intravenous injection of phenylephrine or sodium nitroprusside. Baroreflex was estimated by sigmoidal curve fitting of HR responses. An increased baroreflex gain was observed in chronic DPAG-lesion rats compared to both DPAG-sham (p < 0.01) and SC-lesion (p < 0.05) chronic groups. The chronic DPAG-lesion group showed also an elevation of both the tachycardia (p < 0.05) and bradycardia (p < 0.01) plateaus compared to chronic DPAG-sham rats, while the SC-lesion group showed an elevation of the bradycardia plateau only (p < 0.01). Similar results on baroreflex function were observed following acute lesion of the DPAG, i.e. an increase in baroreflex gain (p < 0.01) and the elevation of both tachycardia (p < 0.05) and bradycardia plateaus (p < 0.01) compared to the acute DPAG-sham group. Resting AP and HR did not differ among the chronic groups. In contrast, the acute lesion of the DPAG produced a reduction in AP (p < 0.01) accompanied by an increase in HR (p < 0.01). The present data suggest that the DPAG is involved in the tonic and reflex control of AP and HR in conscious rats. In addition, the SC seems to contribute to the baroreflex cardioinhibition.