Effects of antisense oligodeoxynucleotide targeting of the alpha(2B)-adrenergic receptor messenger RNA in the central nervous system.

The results of previous studies with genetically engineered mice have suggested that an intact central alpha(2B)-adrenergic receptor (alpha(2B)-AR) subtype mediates the development and maintenance of salt-induced hypertension. In the present study, we sought to further define the role of this receptor by injecting antisense oligodeoxynucleotides (AS-ODNs), targeting a selected sequence of the alpha(2B)-AR mRNA, into the lateral cerebral ventricle of rats that had undergone prior subtotal nephrectomy and dietary salt loading. Cell culture studies showed that these AS-ODNs could block alpha(2B)-AR protein generation. Before AS-ODN injection, blood pressure (BP) averaged 133+/-5 mm Hg during the daytime and rose to 165+/-4 mm Hg during the nighttime activity hours (P<0.001 versus baseline average of 120+/-2 mm Hg). The injection of AS-ODNs during the early afternoon prevented the BP rise and was associated with a significant fall in heart rate (from 385+/-12 to 306+/-15 bpm, P<0.05) and symptoms of sedation that lasted for several hours, with a peak at 3 to 6 hours and full recovery by 24 hours. At that time, a second injection produced identical effects in all rats (n=9). Control rats (n=10) that received scrambled ODN injections had no changes in BP or heart rate patterns, and neither group had evidence of neurotoxicity, indicating that these effects are specifically due to translational inhibition of central alpha(2B)-AR. We conclude that a fully functional central alpha(2B)-AR is necessary for the induction of salt-dependent hypertension.

A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice.

Adenosine administration has been reported to lower blood pressure by activating specific membrane receptors. The rat and human heart and aorta have been previously found to express both A2-type adenosine receptors, which activate adenylyl cyclase, and A3 adenosine receptors (A3AR), which inhibit adenylyl cyclase. In the current study, we used A3 adenosine receptor (A3AR) knock-out mice to examine the hypothesis that the relative levels of the A2-type adenosine receptors and A3AR determine the steady-state levels of cAMP in the cells and may affect blood pressure. We found that the A3AR knock-out mice express normal levels of the A1- and A2-type adenosine receptors. In situ hybridization demonstrated that the level of A3AR is high in the vascular smooth muscle layer of aortas derived from wild-type mice, but is not detectable in the knock-out mice. The steady-state level of cAMP is elevated in the aorta and heart of knock-out mice, as compared to wild-type mice, but is not altered in platelets, where A3AR is not expressed naturally. A3AR knock-out mice possess a blood pressure comparable to this in wild-type mice. However, when challenged with adenosine, the knock-out mice display a further increase in cAMP levels in the heart and vascular smooth muscle and a significant decrease in blood pressure, as compared to wild-type mice. In contrast, the effect of adenosine on ADP-induced platelet aggregation is similar in both types of mice. These studies indicate that the A3AR affects the steady-state level of cAMP in the tissues where it is naturally expressed, and that it influences the blood pressure in response to adenosine.

Genetic predisposition to stroke in relatives of hypertensives.

BACKGROUND AND PURPOSE: The genetic basis of stroke is poorly understood. We evaluated patterns of familial aggregation of hypertension and stroke to test the hypothesis that inherited susceptibility to these disorders may be determined by a common set of factors. METHODS: Genealogical and medical history information was obtained for a cohort of 354 hypertensive probands ascertained in a clinic-based setting, their 1427 first-degree relatives, and 239 of their spouses. Risks of stroke and hypertension in biological and nonbiological relatives were compared with the logistic model of the generalized estimating equations adjusted for age and sex. RESULTS: The risk of hypertension was higher for the parents and siblings of the probands than for spouses (odds ratio [OR]=2.4; 95% CI, 1.8 to 3.4; OR=2.2; 95% CI, 1.6 to 3.0, respectively). When the spouses were used as a reference group, the risk of stroke for parents of the hypertensive probands was 7.3 times higher (OR=7.3; 95% CI, 3.6 to 14.8), while a nonsignificant but slightly increased risk for siblings (OR=1.6; 95% CI, 0.8 to 3.3) was observed. Controlling for hypertension, obesity, smoking, coronary heart disease, diabetes, and cholesterol resulted in decreased estimates of the risk of stroke for parents and siblings (OR(parents)=5.4; 95% CI, 2.6 to 11.2; OR(siblings)=1.2; 95% CI, 0.6 to 2.5). The risk of stroke was significantly higher for hypertensive parents and siblings than for nonhypertensive parents (OR=5.2; 95% CI, 2.8 to 9. 7) and siblings (OR=5.8; 95% CI, 2.1 to 15.9). A history of hypertension was not associated with an increased risk for stroke in spouses (OR=0.7; 95% CI, 0.2 to 3.1). The risk of stroke in hypertensive relatives of probands with stroke was higher than that of the normotensive relatives (OR=13.4). A less elevated risk ratio was observed in the relatives of probands who did not have a stroke (OR=4.0). CONCLUSIONS: Our data showing a higher occurrence of hypertension and stroke in parents of hypertensive probands compared with spouses suggest that some of the genetic factors predisposing to these conditions may be the same. The slightly increased risk to siblings compared with spouses was not significant, suggesting that elucidation of these factors through family studies of stroke may be difficult because of secular trends toward improved treatment for hypertension. Although a history of hypertension increases the risk of stroke among parents and siblings, multivariate analyses revealed a familial component to stroke independent of hypertension.

Chronic sympathetic suppression in the treatment of chronic congestive heart failure.

Previous short-term studies demonstrated that treatment with clonidine produced significant hemodynamic improvement in patients with congestive heart failure (CHF). In this study we followed 12 CHF patients (10 M, 2 F age 63+/-11, 10 with ischemic cardiomyopathy and 2 with dilated cardiomyopathy) treated with 0.15 or 0.075 mg oral clonidine twice daily for 13+/-5 months (range 6-23). with functional evaluation at baseline, 6 weeks and 6 months. There was suppression of circulating catecholamines, associated with significant ameliorations in NYHA class, in duration of exercise tolerance (from 246+/-68 sec to 362+/-30 and 459+/-70 sec, respectively p < 0.02), in ejection fraction (from 32+/-7% to 35+/-5 and 39+/-7% p < 0.04) and in left ventricular enlargement as assessed echocardiographically. There were also improvements in a number of electrophysiologic parameters calculated by computerized analysis of ambulatory ECG tapes, such as heart rate variability, indicating diminished propensity to malignant arrhythmias, as confirmed by decreases in the numbers of isolated premature ventricular contractions, couplets and episodes of non-sustained ventricular tachycardia. The data suggest that chronic central sympathetic suppression with clonidine in CHF results in significant functional amelioration and improved electrophysiologic stability.

Evidence for cell-specific regulation of transcription of the rat alpha2A-adrenergic receptor gene.

We investigated the transcriptional activity of the -131 to -92 region of the rat alpha2A-adrenergic receptor gene. In HT29 cells, this region has a positive effect on transcription, whereas in RINm5F cells, this region has a negative effect on transcription. The -131 to -92 region has a GC box (GGGGCGG) surrounded by overlapping GGAGG repeats. To analyze nuclear factor binding to this region, we made a series of sequence substitutions in the GGAGG repeats, the GC box, or both regions. Gel mobility shift assays indicated that most of the nuclear factor complexes formed between the wild-type -131/-92 sequence and either HT29 or RINm5F extracts were specific for SP1 or related proteins that recognize a GC box. Mutation of either the GGAGG repeats or the GC box did not eliminate the binding of Sp1 or related nuclear factors, suggesting that both the GGAGG repeats and the GC box could bind Sp1-related factors. Mutation of both these sites eliminated the binding of Sp1-related factors. In the absence of SP1 binding sites, this region had a negative effect on transcription in HT29 and a positive effect on transcription in RINm5F cells. These data support the notion that Sp1 and/or a related factor may control both positive and negative gene expression and suggest that the -131/-92 region may be involved in regulating tissue-specific levels of alpha2A-adrenergic receptor gene expression.

A negative regulatory element in the promoter region of the rat alpha 2A-adrenergic receptor gene overlaps an SP1 consensus binding site.

Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.

Alpha 2 adrenergic receptor subtypes expressed in Chinese hamster ovary cells activate differentially mitogen-activated protein kinase by a p21ras independent pathway.

Epinephrine stimulation of rat alpha 2D, alpha 2B, and alpha 2C adrenergic receptor subtypes, expressed stably in Chinese hamster ovary (CHO) cells, caused a rapid, transient activation of mitogen-activated protein kinase (MAPK), with subtype-specific different efficiencies. The order of activation was CHO-2B approximately CHO-2D much greater than CHO-2C. Pertussis toxin blocked the stimulation of MAPK enzymatic activity and the parallel MAPK phosphorylation, demonstrating that these responses are mediated by pertussis toxin-sensitive Gi proteins. Contrary to what has been reported for the alpha 2A subtype expressed in rat-1 fibroblasts, epinephrine did not cause any detectable activation of p21ras in the CHO transfectants. Furthermore, combined application of epinephrine and phorbol myristate acetate had a potent cooperative but not additive effect in clones CHO-2D and CHO-2B but not in CHO-2C, suggesting that protein kinase C is probably differently involved in the signaling by the three alpha 2 receptor subtypes. These results show that in CHO cells, the different alpha 2 adrenergic receptor subtypes utilize differential pathways to activate MAPK in a p21ras-independent way.

Promoter region of the human alpha 2A adrenergic receptor gene.

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.

Pressor systems in hypertension and congestive heart failure. Role of vasopressin.

Elevated peripheral vascular resistance, which characterizes hypertension and congestive heart failure (the latter regardless of absolute blood pressure level) is maintained to a large extent by the combined effects of three major neurohormonal pressor mechanisms: the renin-angiotensin system, the sympathoadrenal system, and arginine vasopressin. Blockade of one of these mechanisms may lead to compensatory stimulation of the others, thus offsetting in part the hemodynamic benefits of a specific intervention. Combination therapy, designed to attack all three systems (with use of an angiotensin converting enzyme inhibitor, a sympathetic blocker such as clonidine, and an antagonist of the vasopressor action of vasopressin), may help in the treatment of such cases. To illustrate this strategy, two experimental studies, one case of malignant hypertension, and one case of congestive heart failure are presented.

Atrial natriuretic peptide inhibits the agonist-induced increase in extent of myosin light chain phosphorylation in aortic smooth muscle.

The effect of atrial natriuretic peptide (ANP) on angiotensin II- and histamine-induced contraction and muscle light chain phosphorylation was examined in strips of rabbit aorta smooth muscle. Preincubation of strips with 10(-7) M ANP prior to addition of either agonist inhibits both the increase in extent of myosin light chain phosphorylation and the contractile response to either 5 x 10(-8) M angiotensin II or 10(-5) M histamine without inhibiting the agonist-induced increase in the intracellular free Ca2+ concentration. Furthermore, in muscle strips precontracted with either angiotensin II or histamine, addition of ANP leads to a prompt relaxation and a prompt decrease in the extent of myosin light chain phosphorylation. These data argue that ANP uncouples the initial agonist-induced Ca2+ transient from the increase in extent of myosin light chain phosphorylation either by inhibiting the Ca2+-dependent activation of myosin light chain kinase or stimulating the activity of a phosphoprotein phosphatase capable of bringing about the rapid dephosphorylation of phosphorylated myosin light chains.

Muscle glycogenolysis during exercise: dual control by epinephrine and contractions.

The interaction of epinephrine and contractions on muscle metabolism was studied in the isolated perfused rat hindquarter. Subtetanic contractions (180/min) through 20 min elicited glycogenolysis and increased phosphorylase a activity. In the soleus, a slow-twitch red muscle, these effects were transient, but when epinephrine at a physiological concentration (2.4 X 10(-8) M) was added to the perfusate, glycogenolysis and phosphorylase activity were sustained throughout contractions. At this high frequency of contractions, the effect of epinephrine was much smaller in the fast-twitch red fibers and not significant in the fast-twitch white fibers of the gastrocnemius muscle. However, during less frequent contractions (30/min) epinephrine increased glycogenolysis and phosphorylase a activity in fast-twitch muscle. The data suggest that epinephrine and muscle contractions exert a dual control of muscle glycogenolysis during exercise: contractions principally stimulate glycogenolysis early in exercise, and a direct effect of epinephrine on muscle is needed for continued glycogenolysis. In addition, epinephrine increased oxygen consumption and glucose uptake in both resting and electrically stimulated hindquarters and, under some conditions, it had a positive inotropic effect on contracting muscle.

Is elevated plasma renin activity of prognostic importance in progressive systemic sclerosis?

The pathologic lesions of the kidney in scleroderma in many respects resemble those of malignant hypertension, perhaps even in the absence of comparable blood pressure elevation. Because the malignant vascular changes have been related to hyperreninemia, we measured plasma renin activity in 23 patients with scleroderma with or without hypertension and/or renal failure. We found that high renin levels in most cases shortly preceded or coincided with a phase of sudden deterioration of the disease, characterized by a rapidly progressive renal failure. The outcome of this phase was invariably fatal, except for two patients in whom bilateral nephrectomy successfully arrested the rapid downhill course. These findings suggest that an unexplained increase in circulating renin levels in an otherwise stable patient with scleroderma may be taken as a possible marker of imminent deterioration requiring close monitoring and immediate therapeutic intervention.

Blood viscosity, plasma proteins, and Raynaud syndrome.

The rheology of the blood was studied in 20 patients with Raynaud syndrome. Sixteen patients had scleroderma, two had nonspecific angiitis, one had systemic lupus erythematosus, and one had Raynaud disease. Viscosity measurements were performed on whole blood, plasma, and suspensions of 45% red blood cells (RBCs). In autologous plasma, over a wide range of shear rates. The relative viscosity, an index of RBC aggregation, was obtained by dividing the RBC viscosity in autologous plasma (at a hematocrit value of 45%) by the plasma viscosity. Concentrations of the plasma globulins and fibrinogen were also measured. The mean plasma viscosity was significantly (P less than .01) elevated over established normal controls. The mean RBC viscosity and the relative viscosity were significantly (P less than .01) elevated over normal controls, as were fibrinogen and the globulins. These studies demonstrate increased blood viscosity and red blood cell aggregation, which may constitute an important hindrance to flow.

The use of angiotensin-converting enzyme inhibitor in the diagnosis and treatment of hypertension.

1. The anti-hypertensive effect of converting enzyme inhibition was evaluated in twenty-three hypertensive patients (seven renovascular, four essential, four malignant, one scleroderma, three chronic renal failure, four primary or idiopathic aldosteronism). 2. In sixteen patients a single injection (1--4 mg/kg) of the inhibitor produced an immediate anti-hypertensive effect, which lasted up to 16 h. In six patients the anti-hypertensive effect of the inhibitor was significantly improved after sodium depletion. 3. Plasma renin activities increased and plasma aldosterone concentrations decreased consistently except in idiopathic aldosteronism. 4. Converting enzyme inhibition provides a direct way of defining the degree of renin-dependency of the hypertension. Accordingly, it can be used diagnostically and for planning appropriate therapy. Therapeutically, it could be advantageous in hypertensive emergencies because of its safety, specificity and capacity to reduce aldosterone secretion.

Angiotensin- and norepinephrine-induced myocardial lesions: experimental and clinical studies in rabbits and man.

The ability of large doses of exogenous angiotensin II to cause widespread multifocal microscopic myocardial necrosis in the rabbit has been confirmed. Angiotensin II also consistently produced acute renal failure with, less consistently, renal tubular necrosis. Norepinephrine infusions caused histologically indistinguishable myocardial lesions, but did not detectably affect renal function or histology. Severe renal failure, induced by bilateral nephrectomy (with or without concurrent glycerol administration) was not associated with similar cardiac lesions. Acute renal failure of comparable or greater severity to that induced by angiotensin II was produced by intramuscular cephaloridine, and was not associated with cardiac lesions. Rabbits infused with saline intravenously or "sham"-operated by simply opening and closing the peritoneal cavity did not develop renal failure and showed no cardiac or renal lesions histologically. Myocardial lesions, apparently identical to those seen in the rabbits, were observed postmortem in three patients known to have had high circulating levels of angiotensin II before death, although in all three cases alternative explanations are possible. Unexplained arrhythmia, cardiac arrest, and central chest pain without clear cardiographic or serum enzyme evidence of myocardial infarction occurred in two other subjects with very high plasma levels of angiotensin II. These attacks ceased after bilateral nephrectomy and a consequent fall in plasma angiotensin II. The cardiac attacks in these five patients all occurred during or shortly after procedures, such as sodium-depleting dialysis, renal artery surgery, or diazoxide administration, known to cause increase in plasma concentrations of renin and angiotensin II.

Malignant hypertension resulting from deoxycorticosterone acetate and salt excess: role of renin and sodium in vascular changes.

The evolution of malignant hypertension was studied under metabolic balance conditions in 11 uninephrectomized rats given deoxycorticosterone acetate and 1% NaCl as drinking water. Changes in sodium and potassium balance were related to changes in blood pressure, plasma renin activity, hematocrit, and kidney histology. After 3-4 weeks of steadily positive sodium balance accompanied by continuously increasing blood pressure up to 185 plus or minus 19 (SE) mm Hg, periods of sodium loss accompanied by evidence of hemoconcentration were observed marking the onset of the malignant phase as defined by the development of fibrinoid necrosis in the kidney. Plasma renin activity remained markedly suppressed both at the fourth week (0.33 plus or minus 0.02 ng/ml hour-1) when the sodium balance was positive and the kidney biopsy negative and at the end of the experiment (0.35 plus or minus 0.36 ng/ml hour-1) when the sodium balance was negative and the kidney histology revealed malignant vasculitis. Infusion of the angiotensin II inhibitor 1-Sar-8-Ala-angiotensin II consistently failed to affect blood pressure, and the kidney tissue norepinephrine level was reduced (0.054 plus or minus 0.01 mug/g) compared with the control level (0.132 plus or minus 0.02 mug/g). We conclude that malignant vasculitis in this model is preceded by hypertension associated with sodium and water retention and is accompanied by negative sodium balance, decreases in body weight, falling blood pressure, and hemoconcentration without demonstrable participation of the renin-angiotensin system or the renal catecholamines.