Linkage between alpha(1) adrenergic receptor and the Jak/STAT signaling pathway in vascular smooth muscle cells.

The Jak/STAT pathway is activated following stimulation of the type I angiotensin II receptor. To examine whether this pathway is shared among other G-protein-coupled receptors, we studied the linkage between the alpha(1) adrenergic receptor and this pathway. The alpha(1) agonist phenylephrine induced tyrosine phosphorylation of Jak2, Tyk2, and STAT1 in vascular smooth muscle cells. The phosphorylation of Jak2 was prevented by the alpha(1) receptor antagonists prazosin and chloroethylclonidine, but not by WB4101, and that of STAT1 was inhibited by prazosin and the Jak2 inhibitor AG490. After stimulation with phenylephrine, Jak2 and STAT1 were found to associate with alpha(1B) receptor. Phenylephrine stimulated the DNA binding activity of STAT1. Protein synthesis promoted by phenylephrine was inhibited by prazosin, AG490, and the introduction of a decoy oligonucleotide for STAT1. These results suggested that alpha(1) receptor is linked to the Jak/STAT pathway and that this pathway mediates alpha(1) agonist-induced smooth muscle hypertrophy.

Ouabain-like immunoreactivity in the medulla oblongata of rats.

An isomer of ouabain, the ouabain-like compound (OI,C), may participate in the regulation of body fluid volume and vascular tone. Forebrain regions, especially the hypothalamus, are reported to be sites of OLC action in the central nervous system. The medulla oblongata is another critical area involved in central cardiovascular regulation. We reported that the microinjection of either monoclonal antibody to ouabain T8B11 or Fab fragment of digoxin-specific antibody into the rostral ventrolateral medulla significantly decreased mean arterial pressure and renal sympathetic nerve activity in anesthetized normotensive rats (TERUYA et al.: J. Clin. Invest. 99: 2791-2798, 1997). Using T8B11, we examined the ouabain-like immunoreactivity in the medulla oblongata of normotensive rats. In periodate-lysine-paraformaldehyde fixed tissues, ouabain-like immunoreactive neurons were detected in the nuclei and regions in the medulla oblongata including the ventrolateral medulla, ventromedial medulla, nucleus ambiguus, caudal raphe nuclei, nucleus of solitary tract, and dorsal motor nucleus of the vagus. When an Fab fragment of digoxin-specific antibody was used as a first antibody, the digoxin-like immunoreactive neurons were distributed in almost the same pattern as those observed with the use of T8B11. In the brain fixed with the "three-step" procedure developed by YAMADA et al. (1987), which was used in a previous ouabain immunohistochemical study of the hypothalamus, ouabain-like immunoreactivity in the medulla oblongata was much weaker in intensity and less restricted in distribution than that in the hypothalamus. These findings suggest that ouabain-like immunoreactivities are present in the medulla oblongata with a manner of distribution different from that seen in the hypothalamus. Some ouabain-immunopositive nuclei and regions in the medulla oblongata, especially the rostral ventrolateral medulla, may be other OLC action sites.

Prolonged NOS inhibition in the brain elevates blood pressure in normotensive rats.

Systemic inhibition of nitric oxide synthase (NOS) evokes hypertension, which is enhanced by salt loading, partly via augmented sympathetic activity. We investigated whether inhibition of brain NOS elevates blood pressure (BP) in normotensive rats and, if so, whether the BP elevation is enhanced by salt loading. After a 2-wk low-salt (0.3%) diet, male Sprague-Dawley (SD) rats were divided into four groups. Groups 1 and 2 received a chronic intracerebroventricular infusion of 0.5 mg . kg-1 . day-1 of NG-monomethyl-L-arginine (L-NMMA), and groups 3 and 4 were given artificial cerebrospinal fluid (aCSF). Groups 1 and 3 were placed on a high-salt (8%) diet, whereas groups 2 and 4 were on a low-salt diet. On day 9 or 10, group 1 showed significantly higher mean arterial pressure (MAP) in a conscious unrestrained state (129 +/- 3 mmHg vs. 114 +/- 3, 113 +/- 1, and 108 +/- 3 mmHg in groups 2, 3, and 4, respectively, P < 0.05). On a high-salt diet, response of renal sympathetic nerve activity but not of BP to air-jet stress was significantly larger in rats given L-NMMA than in rats given aCSF (29 +/- 4% vs. 19 +/- 3%, P < 0.05). When the intracerebroventricular infusions were continued for 3 wk, MAP was significantly higher in rats given L-NMMA than in rats given aCSF irrespective of salt intake, although the difference was approximately 7 mmHg. Thus chronic inhibition of NOS in the brain only slightly elevates BP in SD rats. Salt loading causes a more rapid rise in BP. The mechanisms of the BP elevation and its acceleration by salt loading remain to be elucidated.

Contribution of alpha 2-adrenoceptors in caudal ventrolateral medulla to cardiovascular regulation in rat.

The inhibitory action of alpha 2-agonists on the cardiovascular neurons has been elucidated in the rostral ventrolateral medulla (RVLM) but not in the caudal ventrolateral medulla (CVLM). Our study aimed to clarify whether microinjection of clonidine into the CVLM elicits any cardiovascular effect and whether endogenous alpha 2-adrenoceptor-mediated mechanisms contribute to the tonic activity of the CVLM neurons. In male Sprague-Dawley rats (7-9 wk old, 270-320 g) anesthetized with urethan, unilateral microinjection of 8 nmol of clonidine into the CVLM (n = 10) increased mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) by 12.1 +/- 1.8 mmHg (mean +/- SE, P < 0.01) and 25.8 +/- 4.8% (P < 0.01), while heart rate (HR) remained unaltered. Unilateral microinjection of 2 nmol of SKF-86466, a selective blocker of the alpha 2-adrenoceptors, into the CVLM (n = 10) decreased MAP, HR, and RSNA (-11.6 +/- 2.6 mmHg, -26 +/- 7 beats/min, and -15.3 +/- 1.7%, respectively, P < 0.01 for each). Artificial cerebrospinal fluid caused neither a cardiovascular effect nor a sympathetic response. Prior injection of SKF-86466 into the ipsilateral CVLM attenuated the effects of clonidine. Bilateral microinjection of muscimol into the RVLM abolished the effects of both clonidine and SKF-86466 injected into the CVLM. The pressor and sympathoexcitatory effects of clonidine injected into the CVLM suggest a neuroinhibitory action of the drug on the CVLM neurons. In addition, the depressor and sympathoinhibitory effects of SKF-86466 injected into the CVLM indicated that activation of alpha 2-adrenoceptors by endogenous ligand inhibits CVLM neurons. The effects of clonidine and the alpha 2-adrenoceptor antagonist in the CVLM require the integrity of the RVLM.

Role of ouabain-like compound in the rostral ventrolateral medulla in rats.

To determine whether ouabain-like compound (OLC) exerts modulatory influences on the activity of vasomotor neurons in the rostral ventrolateral medulla (RVLM), we examined the effects of microinjecting ouabain, digoxin-specific antibody Fab fragments, and mAb against ouabain on the rat RVLM. Microinjection of ouabain into the unilateral RVLM of anesthetized normotensive rats elicited dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). The pressor and sympathoexcitatory effects of ouabain in the RVLM were reversed by microinjections of an M2 muscarinic antagonist, gallamine, or digoxin-specific antibody Fab fragments. Furthermore, a prior microinjection in the RVLM of gallamine, digoxinspecific antibody Fab fragments, or kainic acid or intravenous injection of hexamethonium all prevented the pressor and sympathoexcitatory effects induced by a subsequent microinjection of ouabain. Microinjections of either digoxinspecific antibody Fab fragments or gallamine per se significantly decreased baseline MAP and RSNA. Injection of digoxin-specific antibody Fab fragments attenuated the effects of a subsequent injection of gallamine. Microinjection of mAb against ouabain, but not nonspecific IgG, also significantly decreased baseline MAP and RSNA. These results suggest that OLC in the RVLM contributes to the tonic activity of vasomotor neurons in anesthetized normotensive rats, and the action of OLC in the RVLM is at least partly mediated by M2 muscarinic mechanisms.

Brain angiotensin and circulatory control.

1. Components of the renin-angiotensin system (RAS) are found in the brain; both outside and inside the blood-brain barrier. 2. Almost all of the classical actions of the brain RAS are attributable to angiotensin (Ang) II and mediated by AT1 receptors. 3. Circumventricular organs (CVO), which lack the blood-brain barrier, are rich in AngII receptors and monitor circulating AngII levels. In vivo binding studies suggest that the CVO are also accessible to cerebrospinal fluid-derived AngII. 4. The median preoptic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, nucleus tractus solitarius and ventrolateral medulla are inside the blood-brain barrier and are sites of action of brain AngII. In these nuclei, AngII seems to act as an excitatory neurotransmitter or neuromodulator. 5. Actions of AngII in the brain, both inside and outside the blood-brain barrier, are implicated in the central regulation of blood pressure and sympathetic outflow, release of hypothalamic and pituitary hormones and renal sodium handling. 6. Alterations in the activity of brain AngII may be involved in the mechanisms of some types of hypertension.

Brain angiotensin II contributes to the development of hypertension in Dahl-Iwai salt-sensitive rats.

OBJECTIVE: To examine the role of brain angiotensin II in the development of salt-induced hypertension in Dahl-Iwai salt-sensitive (DIS) rats. METHODS: Male DIS and Dahl-Iwai salt-resistant (DIR) rats aged 5 or 6 weeks were implanted with an intracerebroventricular cannula, and either chronic intracerebroventricular infusion of 5 micrograms/day CV-11974, a non-peptide type-1 angiotensin II receptor antagonist or artificial cerebrospinal fluid (aCSF) was started. The rats were fed a diet containing 8% sodium chloride. RESULTS: On day 11 or 12 of chronic infusion, DIS rats given CV-11974 intracerebroventricularly exhibited a significantly lower mean arterial pressure than DIS rats given aCSF intracerebroventricularly or intravenous infusion of CV-11974. In DIR rats, intracerebroventricular infusion of CV-11974 did not alter the mean arterial pressure. Sodium and water balances were similar in all of the groups. Plasma vasopressin and noradrenaline levels did not differ among the groups, although the plasma renin concentration was significantly lower in DIS rats given aCSF intracerebroventricularly. Arterial baroreflex control of heart rate and pressor response to intravenous injection of phenylephrine were not altered in rats given CV-11974 intracerebroventricularly. CONCLUSION: The integrity of the brain renin-angiotensin system is necessary for the development of salt-induced hypertension in DIS rats.

Modulation of baroreflex function by angiotensin II endogenous to the caudal ventrolateral medulla.

Neurons in the ventrolateral medulla (VLM) mainly determine the tonic sympathetic activity. The caudal VLM (CVLM) relays baroreflex signals to the rostral VLM. We have reported that endogenous angiotensin II (ANG II) contributes to the ongoing activity of the VLM neurons. In the present study, we examined if ANG II endogenous to the CVLM modulates the baroreflex function in anesthetized normotensive Sprague-Dawley rats. Changes in renal sympathetic nerve activity (RSNA) in response to changes in mean arterial pressure (MAP) induced by i.v. infusion of phenylephrine and nitroglycerin were recorded before and after bilateral microinjection of [Sar1, Thr8]-ANG II, an ANG II antagonist, into the CVLM. The ANG II antagonist injection into the CVLM significantly increased MAP and RSNA by 17.6 +/- 8.0 mmHg (mean +/- S.D.) and 36.3 +/- 18.1%, respectively. It also significantly increased the baroreflex sensitivity (BS) from -0.49 +/- 0.38 to -0.74 +/- 0.37%/mmHg during nitroglycerin infusion. In contrast, the BS examined by phenylephrine infusion was not altered by the pretreatment with ANG II antagonist. Injection of artificial CSF affected neither the baseline values of MAP and RSNA nor the BS. These results suggest that ANG II endogenous to the CVLM exert a modulating role in baroreflex control of RSNA.

Short-term effects of angiotensin II blockade on renal blood flow and sympathetic activity in awake rats.

To investigate the effects of an angiotensin II type 1 receptor antagonist (CV-11974) on renal blood flow and renal sympathetic nerve activity compared with a calcium antagonist (nicardipine), we measured both parameters in conscious spontaneously hypertensive rats aged 13 to 15 weeks. One to 2 days after surgery, CV-11974 (n = 9) and nicardipine (n = 8) were intravenously administered to decrease arterial pressure in a similar time course and degree of hypotension. CV-11974 increased renal blood flow by 23 +/- 4% at the maximal fall in mean arterial pressure (-32 +/- 1 mm Hg), and renal nerve activity increased by 70 +/- 7%. The maximal increase in renal blood flow (+27 +/- 4%) was observed when mean pressure was reduced by approximately 20 mm Hg. The maximal reduction of renal vascular resistance (-33 +/- 3%) correlated significantly with pretreatment levels of plasma renin concentration (r = -.792). In contrast, nicardipine produced a progressive reduction of renal blood flow and marked increases in heart rate and renal nerve activity. Increases in heart rate and nerve activity were greater than those with CV-11974 treatment (P < .001). At the maximal fall in mean pressure (-32 +/- 1 mm Hg), renal blood flow decreased by 23 +/- 4%, which was significantly correlated with percent changes in renal nerve activity (+150 +/- 11%, r = -.744). Renal denervation in another set of rats (n = 6) improved renal blood flow and renal vascular resistance responses to nicardipine.(ABSTRACT TRUNCATED AT 250 WORDS)

[Continuous measurement of cardiac output during ergometer exercise using an ultrasonic pulsed Doppler flowmeter].

A method for determining cardiac output was developed, which provided the product of the absolute blood flow rate at the aortic arch (as measured non-invasively from the suprasternal notch by an ultrasonic pulsed Doppler flowmeter: UPDF) and the cross-sectional area of the aorta at that level, as estimated from two-dimensional echograms. Using this method, non-invasive continuous measurements of cardiac output were made during bicycle ergometer exercise. The results were summarized as follows: Resting cardiac output values obtained by this method correlated reasonably well with those by the thermodilution method (correlation coefficient r = 0.76). Eight healthy male volunteers exercised for 2 min on a bicycle ergometer at 250 kpm/min, and serial cardiac outputs were determined by this method for five of these subjects during and after the loading. The cardiac output curves thus obtained clearly demonstrated abrupt increases in cardiac outputs initially, followed by rapid returns to resting cardiac output levels after the end of exercise. We intend to conduct additional studies for larger groups of subjects to further evaluate and to establish this method for use in cardiac examinations.

Non-invasive quantitative evaluation of aortic regurgitation using an ultrasonic pulsed Doppler flowmeters.

The severity of aortic regurgitation (AR) is usually evaluated using cineaortography, but this procedure cannot be carried out easily because of its invasive nature. For estimating the severity of AR non-invasively as well as quantitatively, we measured the blood flow in the aortic arch using an ultrasonic pulsed Doppler flowmeter (UPDF) from a suprasternal notch. The rugurgitant ratio was calculated from the waves of relative flow volume and compared with the severity determined cineaortographically. The following results were obtained: 1) Reproducible waves of the aortic arch flow were recorded in all normal subjects and also in 19 out of 23 patients with AR. 2) Distinctive waves of the regurgitant flow, which could not be seen in normal subjects, were recorded in AR except for some mild cases. 3) The regurgitant ratio obtained from the UPDF corresponded well with the severity based on the cineaortogram. It may be concluded that the UPDF is clinically useful and reliable for quantifying AR non-invasively.

[Quantitative cineangiographic evaluation of the valvular regurgitant flow ratio by digital image processing technique (author's transl)].

By application of digital image processing technique to cineangiogram, possibility to calculate the valvular regurgitant flow ratio, especially in patents with aortic regurgitation, was examined. The image processor used was RT6404 made by Amast Computer CO, and the microcomputer to control the image processor was H68TR made by Hitachi Industrial Co. The image of the cineangiogram was photographed by televicamera, and the image processor converted the televi-signal to digitized image. Total volume and concentration of contrast medium in the ventricle was integrated in each systole and diastole. In patients without aortic regurgitation, the volume of contrast medium was not increased in the next diastole after the end of injection. On the contrary, in cases of aortic regurgitation, the volume of contrast medium was increased in the next diastole because of regurgitation. Thus, the regurgitant flow ratio could be calculated from the ejected volume in systole immediately before this diastole and the increased volume caused by the regurgitation.

[Non-invasive quantitative evaluation of aortic regurgitation by ultrasonic pulsed Doppler flowmeter (author's transl)].

A usefulness of non-invasive and quantitative evaluation of aortic regurgitation (AR) was investigated by measuring the aortic arch flow from the suprasternal notch with ultrasonic pulsed Doppler flowmeter (UPDF). Measurement of the blood flow was made on 19 patients with AR, calculating the regurgitant ratio from the waves of relative flow volume, and the estimated grade of AR was compared with the severity judged from the cineaortogram. The results were as follows: 1) The aortic arch flow was recorded in 17 out of 19 patients with AR. 2) Except mild cases, the diastolic regurgitant flow was recorded in patients with AR. 3) The regurgitant ratio obtained by the UPDF correlated well with the grade of the severity of AR judged from the cineaortogram.