Warren Lockette

α2-Adrenergic receptor gene polymorphism and hypertension in blacks

alpha 2-Adrenergic receptors are found on presynaptic neurons of the central and peripheral nervous systems, on blood vessels, on platelets, on adipocytes, and in the kidney and pancreas. Activation of these ubiquitous adrenoreceptors results in decreased neuronal norepinephrine release, vasodilation, a fall in blood pressure, platelet aggregation, increased sodium excretion, and decreased insulin release. We hypothesized that defects in alpha 2-adrenergic receptors, or postreceptor defects, could explain the increased prevalence of hypertension in blacks. To test our hypothesis, we first determined whether or not a polymorphism of the alpha 2-adrenergic receptor gene was associated with pathologic elevations in blood pressure in American blacks. Dra-I identified a restriction fragment-length polymorphism (RFLP) of 6.3 and 6.7 kb of the alpha 2-adrenergic receptor gene on chromosome 10 in humans. Of 227 patients studied, 13/107 hypertensive subjects were homozygous for the 6.3-kb allele, whereas only 3/120 normotensive volunteers were homozygotes (P = .008). When analyzed by race, 13/82 black hypertensive subjects were homozygous for the 6.3-kb allele, whereas only 2/59 normotensive blacks were homozygous for the 6.3-kb alleles (P = .02). However, only 1/61 white normotensive and 0/25 white hypertensive subjects were homozygous for the 6.3-kb allele (P = 1.00). Ethnic variation among blacks may explain our findings. Alternatively, a genetic polymorphism in, or near, the alpha 2-adrenergic receptor on chromosome 10 can contribute to the development of hypertension in blacks.

Genetic polymorphism of the α2-adrenergic receptor is associated with increased platelet aggregation, baroreceptor sensitivity, and salt excretion in normotensive humans

It is likely that a number of independent heritable traits, each encoded by a singular gene, contribute to pathologic elevations in blood pressure in humans. Genetic polymorphisms of individual genes may result in intermediate phenotypes which, by themselves, do not raise blood pressure, but, coupled with environmental or epistatic forces, contribute to the prevalence of human hypertension. The gene for the alpha 2-adrenergic receptor encoded by chromosome 10 (C10 A2AR) is polymorphic, and Southern blotting with a cDNA probe following restriction enzyme digest of this gene results in fragments of either 6.3 kb or 6.7 kb in size. We reported an association between homozygosity for the 6.3 kb allele and hypertension in blacks. Blacks with hypertension also have an increased risk for thrombotic stroke, increased baroreceptor sensitivity, and decreased sodium excretion. We noted that the C10 A2AR, which modulates norepinephrine release in blood-pressure-regulating regions of the brain, is also expressed on platelets and in the kidney. We postulated that functional changes associated with the C10 A2AR gene polymorphism could be responsible for increased baroreceptor sensitivity, epinephrine-mediated platelet aggregation, and decreased sodium excretion in some individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular reactivity and high dietary eicopentaenoic acid

Epidemiologic studies suggest that high dietary intake of eicosapentaenoic acid (EPA), a precursor of the trienoic prostaglandins, is associated with a low incidence and reduced extent of myocardial infarction. Vascular reactivity of isolated aortic strips from rats maintained for 3 weeks on a control diet or on a diet supplemented with menhaden fish oil (17% EPA) was examined with norepinephrine, sodium arachidonate, KC1, PGF2 alpha and nitroprusside. Aortic strips from rats fed the fish oil diet were significantly less responsive to the contractile effects of norepinephrine and arachidonate compared to those from control diet rats. Treatment of aortic strips with indomethacin decreased responsiveness to norepinephrine. The magnitude of the decrease was greater in control rats resulting in a similar vascular response between the 2 groups after blockade. Contractions to arachidonate were abolished by indomethacin. There were no differences in vascular responses to KC1, PGF2 alpha and nitroprusside in aortic strips from control diet rats and those from the fish oil diet rats. Aortic strips from the fish oil diet rats contained more EPA than those from the control diet rats. Thus, the contractile effect of norepinephrine in isolated rat aortic strips is normally augmented by intrinsic prostaglandins, and this augmentation is diminished by dietary intake of EPA.

Chlorthalidone Decreases Platelet Aggregation and Vascular Permeability and Promotes Angiogenesis

Variations in diuretic-mediated inhibition of carbonic anhydrase-dependent chloride transport in platelets and vascular smooth muscle could account for the contrasting efficacy of the thiazide and thiazide-like diuretics in reducing cardiovascular morbidity in patients with hypertension. We assessed platelet carbonic anhydrase activity and catecholamine-induced platelet aggregation in the presence of a thiazide and a “thiazide-like” inhibitor of the sodium-chloride cotransporter. Individual variation in platelet carbonic anhydrase activity correlated with contrasting sensitivity to epinephrine-mediated platelet aggregation. Both chlorthalidone, which potently inhibits platelet carbonic anhydrase, and bendroflumethiazide, which has much less effect on this enzyme, increased the amount of epinephrine needed to induce platelet aggregation when compared with the absence of a diuretic. However, chlorthalidone was significantly more effective than bendroflumethiazide in reducing epinephrine-mediated platelet aggregation. Chlorthalidone also induced marked changes in the number of gene transcripts for two proteins that mediate angiogenesis and vascular permeability, vascular endothelial growth factor C and transforming growth factor-&bgr;3; chlorthalidone and bendroflumethiazide had contrasting effects on the expression of vascular endothelial growth factor C. Chlorthalidone and bendroflumethiazide reduced vascular permeability to albumin, but only chlorthalidone increased angiogenesis. Thiazides and thiazide-like diuretics can comparably reduce blood pressure, but the drugs in this class are not all alike. It can be suggested from our findings that thiazide and thiazide-like diuretics vary in their pleiotropic effects on platelets and in the vasculature, and these differences could explain the contrasting ability of these drugs to reduce cardiovascular morbidity despite comparable reduction in blood pressure.

Mechanism of Epinephrine-Induced Platelet Aggregation

We report that a genetic polymorphism of the alpha2-adrenergic receptor (A2AR) encoded by chromosome 10 is associated with hypertension and an increase in epinephrine-mediated platelet aggregation in humans. The mechanism responsible for this heritable contrast in sensitivity to epinephrine is unknown. We tested our hypothesis that epinephrine-induced platelet aggregation is mediated by activation of chloride transport. We measured epinephrine-mediated increases in optical density of gel-filtered platelets suspended in a bicarbonate-buffered physiological salt solution. Compared with platelets incubated in the control buffer (130 mmol/L NaCl), platelets incubated with either bumetanide, a Na/K/2Cl cotransport inhibitor; anthracene-9-carboxylic acid, a chloride channel blocker; or acetazolamide, an agent that blocks ATP-dependent chloride transport had significantly decreased aggregation responses to epinephrine. When measured fluorometrically, epinephrine significantly increased intraplatelet chloride concentrations. Chloride-dependent modifications of epinephrine-induced platelet aggregation were not attributable to changes in A2AR ligand binding characteristics or to the concentration of platelet cAMP. Finally, subthreshold concentrations of epinephrine also potentiated thrombin-induced platelet aggregation, and blockade of chloride transport diminished this synergistic action of epinephrine on thrombin-stimulated platelet aggregation. Heritable differences in epinephrine-mediated platelet aggregation may be attributable to genetic differences in chloride transport in platelets. Furthermore, because we observed a necessary role for chloride transport in epinephrine-mediated platelet aggregation, pharmacological agents that block chloride transport, such as diuretics, may provide salutary protection against vascular thrombosis in patients with hypertension independent of the effect of these drugs on blood pressure.

The Metabolic Syndrome: A Modern Plague Spread by Modern Technology

Malnutrition and infectious disease represent the most common health threats facing the developing world. However, increasing technological developments and the expansion of western culture have contributed to the increasing prevalence of the metabolic syndrome. The epidemiologic significance and potential costs to governmental health care systems of an increasing incidence of metabolic syndrome could become high. The role of environmental influences that lead to the development of the metabolic syndrome needs to be explored. Because the metabolic syndrome becomes more common as nations develop, investigations into the ramifications of this disease often come too late.

Cyclic GMP-Independent Mechanisms of Nitric Oxide-Induced Vasodilation

1. The aim of the present study was to test in vitro if NO acts through a cyclic GMP-independent mechanism to activate Ca2+-dependent potassium channels (K+(Ca)), leading to membrane hyperpolarization and vasodilation in rat tail artery. 2. Acetylcholine and sodium nitroprusside stimulated a significant increase in cyclic GMP (190+/-23 and 180+/-15 pmol/g, respectively) compared with agonist-free conditions (132+/-15 and 130+/-15 pmol/g, respectively); these agonist-mediated increases in cyclic GMP were completely abolished by treatment with the guanylate cyclase inhibitor methylene blue (122+/-10 and 60+/-8 pmol/g, respectively). 3. In contrast, relaxation to acetylcholine (10(-7) mol/l; 61+/-3%) and sodium nitroprusside (10(-8) mol/l; 97+/-1%) were significantly, but not completely, attenuated by methylene blue (30+/-5 and 79+/-3%, respectively); maximum relaxation to sodium nitroprusside (10(-7) mol/l) was unaffected by methylene blue. 4. Depolarization-induced contraction of vessels with KCl inhibited relaxation to both acetylcholine (10(-7) mol/l; 18+/-4%) and sodium nitroprusside (10(-8) mol/l; 57+/-7%). Furthermore, the specific K+(Ca) antagonist charybdotoxin significantly inhibited relaxation to sodium nitroprusside (10(-8) mol/l; 52+/-7%). 5. An additive inhibitory effect on relaxation to sodium nitroprusside (10(-8) mol/l) was observed with a combination of methylene blue and KCl (26+/-6%) or charybdotoxin (34+/-3%). 6. These data suggest that NO stimulates membrane hyperpolarization via K+(Ca) activation, in addition to guanylate cyclase, to cause relaxation in rat tail artery.

Ethnic differences in titratable acid excretion and bone mineralization.

PURPOSE To test our hypothesis that differences in urinary calcium excretion among blacks and whites may be secondary to ethnic variations in acid (H(+)) metabolism and to prove that increases in titratable acid excretion would be found among individuals predisposed to the development of stress fractures. METHODS We administered 8 g NH(4)Cl acutely to 11 black and 18 white healthy volunteers and measured urinary sodium, calcium, and acid excretions. We measured the Na(+)/H(+) antiporter activity using acid-loaded platelets as surrogate markers for this exchanger expressed in renal epithelial cells. We also compared differences in titratable acid excretion among a cohort of subjects with, and without, a history of stress fracture. RESULTS NH(4)Cl-induced increases in titratable urinary acid correlated with changes in the renal excretion of calcium and sodium, and stimulated acid excretion correlated with basal acid loss. Despite comparable changes in plasma pH, whites, when compared to blacks, had much greater basal acid excretion and NH(4)Cl-induced acid excretion. Whites also had much greater baseline calcium excretion rates when compared to blacks. Following acid loading, whites continued to exhibit greater calcium excretion rates than blacks. Acid loading significantly decreased sodium excretion in whites but not in blacks. Blacks also had significantly attenuated Na(+)/H(+) exchange activity. In a cohort of resting, athletic students, we found enhanced basal H(+) and phosphate excretion among subjects who experienced stress fractures during their rigorous physical training when compared to those individuals who did not. CONCLUSION Blacks may have a greater endogenous buffering capacity than whites, or the reported ethnic differences in sodium and calcium excretion rates between blacks and whites may be secondary to racial variations in renal H(+) excretion. We conclude that both ethnic differences in bone mineralization and bone integrity in athletes are mediated by heritable differences in titratable acid excretion.

Effect of the alpha 2-adrenergic antagonist yohimbine on orthostatic tolerance.

We studied the effect of yohimbine, a drug that inhibits presynaptic Oj-adrenergic receptors and increases the neuronal release of norepinephrine from the central and sympathetic nervous systems, on tolerance to cardiovascular stress in 10 untrained, healthy subjects. Using radioligand binding of tritiated yohimbine to platelets, these subjects were found to have a normal complement of a<2-adrenergic receptors (174±18 [±SM] receptors/platelet) with normal X, (1.93±0.17 nmol/1). Lower body negative pressure was used to test responses to cardiovascular stress in the subjects after they received either placebo or 20 mg yohimbine. Graded lower body negative pressure from 0 to −40 mm Hg significantly decreased systolic blood pressure from 116±3.7 to 106±5.8 mm Hg, increased heart rate from 54±3 to 68±7 beats/min, decreased forearm blood flow from 1.8 ±0.21 to 1.36±0.25 ml/100 ml/min, and increased forearm vascular resistance from 55.76±12.1 to 77.26±15.8 mm Hg/ml/min. Yohimbine increased the blood pressure at rest and during lower body negative pressure, but these changes were not significantly different from values recorded from the individuals when they were given placebo. Compared with placebo, however, yohimbine significantly increased forearm blood flow at rest (1.80±0.21 vs. 2.66±OJ1 ml/100 ml/min, p < 0.05) and during −40 mm Hg of lower body negative pressure (U6±0.25 vs. 1.91±0.28 ml/100 ml/min, p < 0.05). We also found that yohimbine significantly increased the plasma insulin concentration in these fasted subjects (9.4±2.4 vs. 14.5±1.4 ng/ml,/?<0.05) without inducing hypoglycemia. Because this agent increases forearm blood flow, yohimbine might be useful in treating the orthostatic hypotension and ischemic vascular disease that results from the autonomic insufficiency common in patients with diabetes mellitus.

Chloride increases adrenergic receptor-mediated platelet and vascular responses.

BACKGROUND We postulated that increasing intracellular chloride concentration ([Cl-]i) in human platelets would potentiate alpha2 adrenergic receptor (A2AR)-mediated platelet aggregation, and that vascular reactivity would also be increased by raising [Cl-]i in blood vessels. We further hypothesized that ligands binding to the A2AR would increase [Cl-]i by stimulating carbonic anhydrase-dependent chloride/bicarbonate exchange. Because diuretics are potent inhibitors of carbonic anhydrase, we speculated that these agents inhibit platelet aggregation and vascular contractility through inhibition of chloride influx by decreasing carbonic anhydrase activity, and subsequently, chloride/bicarbonate exchange. The aim of this study was to test these hypotheses. METHODS Platelet aggregation was measured by determining changes in optical density of platelet-rich plasma. Contractile responses to A2AR agonists were recorded in isolated vascular smooth muscle. The substances [Cl-]i and intracellular pH (pHi) were measured using microfluorometric methods. Carbonic anhydrase activity and chloride/bicarbonate exchange were determined by an in vitro assay based on the Stewart cycle. RESULTS Increasing [Cl-]i potentiated platelet aggregation and vascular contractility, and epinephrine raised [Cl-]i by stimulating carbonic anhydrase-dependent chloride/bicarbonate exchange. Furthermore, diuretic-dependent inhibition of carbonic anhydrase activity decreased chloride/bicarbonate exchange. CONCLUSIONS Our data support the concept that diuretics inhibit carbonic anhydrase activity and chloride/bicarbonate exchange in platelets and vascular smooth muscle. The ensuing reduction in [Cl-]i that is induced by diuretics in these tissues could play a role in reducing the effect of catecholamines on precipitating thrombotic stroke or myocardial infarction.