Sequencing and characterization of the human thiazide-sensitive Na-Cl cotransporter (SLC12A3) gene promoter.

The thiazide-sensitive Na-Cl cotransporter SLC12A3 displays expression restricted to distal convoluted tubule cells where it catalyzes the uptake of sodium and chloride through the apical membrane. We sequenced 1959 bp of the 5' flanking region of human SLC12A3, located the area of transcription initiation, and used deletion constructs of the flanking region to determine areas that affect reporter gene expression in two cell lines, MDCT and CHO. Amplification of the 5' end of SLC12A3 cDNA from an adapter-ligated human kidney cDNA library demonstrated that transcription initiation is confined to an area from -18 to -6 bp upstream of the translation start codon. Maximum promoter activity (9.815 +/- 0.864 times control) was observed in MDCT cells using a promoter containing 1019 bp of the 5' flanking region. A promoter containing only 134 bp of the 5' flanking region upstream of the translation initiation codon maintained reporter gene expression at levels equal to 75% of that maximally observed (7.375 +/- 0.533 times control). Sequence analysis of this minimal promoter responsible for most of the SLC12A3 promoter activity revealed a TATA element, two Sp binding sites, a potential E box, and a potential binding site for NF-1/CTF or NY-I/CP-I. This promoter, and all other promoter constructs from SLC12A3, displayed repressor activity in CHO cells. A construct containing sequence 94 bp upstream of the initiation codon with two potential Sp binding sites was required for this repression. Protein-DNA interactions between the 182 bp region immediately upstream of the start codon and the nuclear proteins from rat kidney cortex and HeLa cells were examined to further clarify the role of the putative binding sites for SLC12A3 expression. Physiological studies investigating the effects of osmolarity, pH, and mineralocorticoid steroid on promoter activity demonstrated that the promoter activity was repressed by acidification, whereas no effects of increased osmolarity or deoxycorticosterone acetate addition were observed.

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.

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.

Alpha 2-adrenergic agonists increase cellular lactate efflux.

We reported previously that genetic polymorphisms of the alpha 2-adrenergic receptor are associated with hyperinsulinemia, diabetes mellitus, and hypertension in blacks. The evolutionary driving force for maintaining such deleterious mutations in the black population is unknown. Recognizing that vascular alpha 2-adrenergic receptors mediate cold-induced vasoconstriction and that temperature maintenance is a primary thrust of cellular metabolism, we postulated that vascular alpha 2-adrenergic receptors contribute significantly to metabolic heat generation in homeotherms such as humans. Using aerobic lactate production as an indicator of thermogenesis, we measured metabolic heat production in HT29 cells that expressed the gene encoding human vascular alpha 2-adrenergic receptors. Epinephrine, an alpha 2-adrenergic receptor agonist, increased net lactate efflux from 226 +/- 20 to 280 +/- 20 nmol/min (mean +/- SE) (P = .06). Clonidine, a more specific alpha 2-adrenergic agonist, increased lactate efflux from 110 +/- 6 to 156 +/- 8 nmol/min (P < .01). Similarly, in the presence of physiological concentrations of glucose (5.5 mmol/L), insulin increased lactate production from 123 +/- 6 to 175 +/- 10 nmol/min (P < .01). Because differences in aerobic glycolysis may also explain the heat intolerance and abnormal fuel homeostasis found in genetically hypertensive rats, we also measured lactate production in cultured vascular smooth muscle cells isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control Wistar-Kyoto rats (WKY). Vascular smooth muscle cells from SHRSP had significantly greater lactate efflux compared with cells from normotensive WKY (296 +/- 4 versus 172 +/- 2 nmol/min, P < .001). These differences were not due to abnormalities in glucose uptake, as lactate efflux was greater in SHRSP cells compared with WKY cells when dextrose was replaced with equimolar concentrations of fructose (230 +/- 6 versus 138 +/- 2 nmol/min, P < .001). alpha 2-Adrenergic agonists increase lactate efflux in HT29 cells, and abnormalities in vascular smooth muscle lactate metabolism in genetically hypertensive rats is independent of altered glucose uptake. These data provide support for our hypothesis that balanced polymorphisms of the alpha 2-adrenergic receptor could offer protection against cold stress by increasing the thermogenic response associated with aerobic lactate production.

Metoclopramide does not inhibit atrial natriuretic factor-stimulated diuresis in man.

BACKGROUND: An understanding of the reflex hormonal responses that occur with a redistribution of the blood volume upon exposure to microgravity or during water immersion is operationally relevant. Further, dopamine receptor antagonists, which are used in the treatment of the motion sickness induced by microgravity, or at sea, may affect fluid metabolism. Atrial natriuretic factor (ANF) is a potent diuretic and natriuretic hormone that is released following central blood volume expansion in man. ANF is also a potent inhibitor of angiotensin- and potassium-stimulated aldosterone release. HYPOTHESIS: Since ANF-induced diuresis may be mediated by dopamine, we sought to determine whether inhibition of dopamine receptors blocks ANF-induced natriuresis and diuresis. Also, since ANF has been shown to inhibit aldosterone secretion induced by adrenocorticotropic hormone (ACTH) in vitro, we investigated whether ANF decreases aldosterone in man infused with exogenous ACTH. METHODS AND RESULTS: In six healthy, sodium-replete men, infusion of synthetic ANF (0.01 microgram.kg.min, Anaritide, Wyeth Laboratories) significantly increased urine flow from 7.1 +/- 0.7 to 11.7 +/- 1.8 ml.min-1 (p < 0.05) and decreased aldosterone from 74.7 +/- 9.0 to 55.8 +/- 6.5 pg.ml-1 (p = N.S.). Metoclopramide (Met), a dopaminergic antagonist, increased plasma aldosterone from 104.5 +/- 8.9 to 163 +/- 12.5 pg.ml-1 (p < 0.05). ANF-induced diuresis was not inhibited by Met, but ANF significantly inhibited Met-stimulated increases in plasma aldosterone. ANF did not attenuate ACTH-stimulated increases in plasma aldosterone. Also, ANF-induced diuresis and natriuresis were not affected by concomitant infusions of ACTH, but the ANF-induced kaliuresis was significantly attenuated by ACTH. CONCLUSION: These studies suggest: a) Synthetic ANF induces a diuresis, natriuresis and kaliuresis; b) the D2 receptor antagonist Met increases plasma aldosterone; c) ANF-induced diuresis is not subject to dopaminergic blockade with Met, but Met-induced increases in plasma aldosterone are inhibited by ANF; and d) ANF does not attenuate ACTH-stimulated increases in mineralocorticoid production. These studies have operational significance, as they demonstrate that D2 dopaminergic blockade by the antinausea agent metoclopramide does not prevent the effects of increased ANF in response to central volume expansion such as occurs during exposure to microgravity or following water immersion.

Genetic polymorphism of the alpha 2-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)

Alpha 2-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.

Controversy in the pharmacologic treatment of hypertension in African Americans.

In 1993, the National Institutes of Health published the recommendations of the fifth, and most recent, US Joint National Committee on the Detection, Evaluation, and Treatment of High Blood Pressure. Many believe that this report advocates the use of inexpensive diuretics and beta-adrenergic-receptor blocking agents as first-line agents in the pharmacologic treatment of hypertension. It has been argued that this recommendation is engendered by primary concerns about costs rather than efficacy or patient satisfaction. The contrary view, however, contends that new, more expensive therapeutic drugs have not demonstrated long-term reductions in morbidity and mortality. For those who train physicians in the treatment of hypertension in African Americans, awareness of this dispute is pivotal so that informed decisions will be made by both the physician and patient.

Frequency of a deletion polymorphism in the gene for angiotensin converting enzyme is increased in African-Americans with hypertension.

In white populations, a deletion polymorphism in the gene for angiotensin converting enzyme (ACE) appears to be associated with increased risk for myocardial infarction but not for hypertension. In a population-association study in African-Americans, we compared the frequency of the ACE deletion polymorphism in subjects with hypertension versus those with normal blood pressure. The frequency of the ACE deletion allele was greater in African-Americans with hypertension than in those with normal blood pressure (P < 0.05). These findings raise the possibility that in some patient subgroups, sequence variation in or near the ACE gene may contribute to the risk for hypertension.

Vasopressin inhibits diuresis induced by water immersion in humans.

We tested the hypothesis that 1-desamino-8-D-arginine vasopressin (DDAVP), a V2-receptor agonist, could inhibit the diuresis induced by water immersion in humans. Water and electrolyte excretion, plasma atrial natriuretic factor concentration, and plasma aldosterone concentration were measured initially and after 3 h of water immersion in 13 healthy sodium-replete men given either placebo or 20 micrograms of intranasal DDAVP. Guanosine 3',5'-cyclic monophosphate and urea excretion and urine osmolality were also determined. DDAVP inhibited the diuresis induced by water immersion in men: 758 +/- 168 (SE) ml/3 h in the placebo group vs. 159 +/- 28 ml/3 h in the DDAVP group (P less than 0.05). After 3 h of water immersion, plasma atrial natriuretic factor concentrations were increased from 11 +/- 2 to 20 +/- 4 pg/ml in the placebo group and from 14 +/- 2 to 33 +/- 4 pg/ml in the DDAVP group (P less than 0.05). Plasma aldosterone concentrations were decreased from 98 +/- 18 to 45 +/- 6 pg/ml in the placebo group (P less than 0.05) and from 54 +/- 17 to 25 +/- 5 pg/ml in the DDAVP group (P less than 0.05). Despite these changes in aldosterone and atrial natriuretic factor concentrations, which should increase sodium excretion, DDAVP decreased the natriuresis induced by water immersion in humans: 56 +/- 8 meq Na+/3 h in the placebo group vs. 36 +/- 6 meq Na+/3 h in the DDAVP group (P less than 0.05). DDAVP may be used to prevent the diuresis associated with central redistribution of blood volumes that occur during water immersion.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered coriolis stress susceptibility in essential hypertension.

Patients with hypertension frequently have vague complaints of dizziness and many other symptoms experienced by healthy individuals with motion sickness. We examined vestibular function in patients with essential hypertension, and we determined whether patients with essential hypertension are more prone to motion sickness using Coriolis stress testing. Vestibular function and Coriolis stress susceptibility were measured in 12 normotensive (NT) and seven asymptomatic patients with mild essential hypertension (HT). The Coriolis stress susceptibility index (CSSI) was calculated from the number of head movements in the four cardinal directions an individual could complete while being rotated in a computerized chair at increasing velocity before they developed motion sickness. The patients with hypertension had normal vestibular function and normal vestibuloocular responses as measured by standard techniques. Subjects with hypertension had significantly decreased Coriolis stress susceptibility scores compared to normotensive subjects (NT, 29.70 +/- 4.8; v HT, 5.48 +/- 2.0, P less than .001) and significantly decreased suppression of postrotatory nystagmus (NT, 44.5% +/- 3.8; v HT, 19.1% +/- 6.9, P less than .05). Medical treatment of hypertension did not result in an increased tolerance to provocative stimuli for motion sickness. It is suggested from our data that an increased susceptibility to motion sickness and abnormal vestibular responses to normal motion may account for many of the vague symptoms of "dizziness" reported by a large number of hypertensive patients.

Atrial natriuretic factor increases vascular permeability.

An increase in central blood volume in microgravity may result in increased plasma levels of atrial natriuretic factor (ANF). Since elevations in plasma ANF are found in clinical syndromes associated with edema, and since space motion sickness induced by microgravity is associated with an increase in central blood volume and facial edema, we determined whether ANF increases capillary permeability to plasma protein. Conscious, bilaterally nephrectomized male rats were infused with either saline, ANF + saline, or hexamethonium + saline over 2 h following bolus injections of 125I-albumin and 14C-dextran of similar molecular size. Blood pressure was monitored and serial determinations of hematocrits were made. Animals infused with 1.0 micrograms.kg-1.min-1 ANF had significantly higher hematocrits than animals infused with saline vehicle. Infusion of ANF increased the extravasation of 125I-albumin, but not 14C-dextran from the intravascular compartment. ANF also induced a depressor response in rats, but the change in blood pressure did not account for changes in capillary permeability to albumin; similar depressor responses induced by hexamethonium were not accompanied by increased extravasation of albumin from the intravascular compartment. ANF may decrease plasma volume by increasing permeability to albumin, and this effect of ANF may account for some of the signs and symptoms of space motion sickness.

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

We studied the effect of yohimbine, a drug that inhibits presynaptic alpha 2-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 alpha 2-adrenergic receptors (174 +/- 18 [+/- SEM] receptors/platelet) with normal Kd (1.93 +/- 0.17 nmol/l). 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 +/- 0.31 ml/100 ml/min, p less than 0.05) and during -40 mm Hg of lower body negative pressure (1.36 +/- 0.25 vs. 1.91 +/- 0.28 ml/100 ml/min, p less than 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, p less than 0.05) without inducing hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of 8-bromo-cyclic guanosine monophosphate on intracellular pH and calcium in vascular smooth muscle.

Endothelium-dependent vasodilators, nitrates, and atrial natriuretic factor relax blood vessels by increasing vascular cyclic guanosine monophosphate (cGMP). The mechanisms by which cGMP relaxes vascular smooth muscle (VSM) are not known. Since contraction of VSM is associated with increased intracellular calcium and pH, we hypothesized that cGMP may decrease vascular tone by lowering ionized, intracellular calcium [( Ca2+]i) and pH. We used microfluorometry to measure cGMP-induced changes in intracellular calcium and pH of cultured A7r5 VSM cells after stimulation with contractile agonists. A cGMP analogue, 8-Br-cGMP, blocked vasopressin- but not thrombin-stimulated increases in [Ca2+]i. High extracellular potassium concentrations [( K+]) increased [Ca2+]i, but the attenuation of [Ca2+]i by 8-Br-cGMP was not statistically significant. 8-Br-cGMP also attenuated vasopressin- but not thrombin-stimulated alkalinization of VSM cells. cGMP may decrease vascular tone by decreasing [Ca2+]i and pH, but these changes are dependent on the contractile agonist studied.

Vascular relaxation and cGMP in hypertension.

Isolated aortas from hypertensive rats have a decreased relaxation response to acetylcholine chloride (ACh), the calcium ionophore A23187, and sodium nitroprusside (SNP). Since the vascular relaxation responses to these vasodilators may be a result of increases in guanosine 3',5'-cyclic monophosphate (cGMP), we measured the cGMP response to these agents in isolated aortas from normotensive rats and rats with either mineralocorticoid-induced hypertension (DOCA), renovascular hypertension (1K1C), or coarctation-induced hypertension (Coarc). The aortas from the hypertensive rats had decreased basal levels of cGMP and attenuated increases in cGMP in response to ACh and A23187. Rises in cGMP in response to SNP were also attenuated in aortas from the hypertensive rats, even at concentrations that induced maximum relaxation of blood vessels from normotensive and hypertensive rats. The relaxation responses to atrial natriuretic factor (ANF) and the cGMP generated in isolated aortas by ANF were attenuated in hypertension. Removal of the endothelium markedly attenuated cGMP generation in response to ANF in vessels from normotensive and Coarc rats, but the relaxation responses to ANF were unaltered in vessels after the removal of the endothelium. The reversal of experimentally induced hypertension was associated with increases in cGMP levels following exposure of the isolated vessels to ACh. Also, vessels treated with methylene blue relaxed in response to SNP despite inhibition of cGMP accumulation. The decreased relaxation response to endothelium-dependent vasodilators is accompanied by decreases in cGMP accumulation; the decreased vascular cGMP content in response to endothelium-dependent vasodilators is not due to increases in phosphodiesterase activity of vascular smooth muscle; and SNP may relax blood vessels through "cGMP-dependent" and "cGMP-independent" mechanisms.

Phorbol ester, vascular relaxation, and cyclic guanosine 3',5'-monophosphate.

The relaxation of phenylephrine-contracted blood vessels by acetylcholine, nitroprusside, or atrial natriuretic factor has been linked to elevations in cyclic guanosine 3',5'-monophosphate (cGMP). Also, 8-bromo-cGMP can induce vascular relaxation in isolated vascular smooth muscle contracted with phenylephrine. We determined whether these cGMP-dependent vasodilators could relax isolated rat aortas contracted with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. cGMP was measured by radioimmunoassay. Acetylcholine, nitroprusside, and atrial natriuretic factor induced relaxation in vascular smooth muscle contracted by 12-O-tetradecanoylphorbol-13-acetate. These relaxation responses were accompanied by elevations of cGMP. However, the sensitivity to these vasodilators was markedly decreased in phorbol ester-contracted vessels compared to phenylephrine-contracted vessels. Nifedipine and superoxide dismutase induced small but significant relaxations in phorbol ester-contracted vessels; however, blood vessels contracted with phenylephrine and phorbol ester relaxed completely with papaverine. There was a marked decrease in sensitivity to 8-bromo-cGMP in phorbol ester-treated vessels compared to phenylephrine-contracted vessels. Contractions induced by phorbol ester were not inhibited by amiloride or chlorpromazine. Also, following incubation in potassium-free salt solution, vessels incubated with phenylephrine or phenylephrine and phorbol ester underwent similar relaxations when exposed to potassium chloride. The contractile state induced by phorbol ester has decreased sensitivity to cGMP-dependent vasodilators. This may be due to nonspecific effects of the phorbol ester or to the mechanism by which protein kinase C activation maintains vascular tone.

Endurance training and human alpha 2-adrenergic receptors on platelets.

Trained endurance athletes have a smaller rise in blood pressure, heart rate, and catecholamine response to stress when compared to an untrained, control population. Since pre-synaptic alpha 2-adrenergic receptors modulate the control of epinephrine release from nerve terminals, and since platelets are used as models of monoaminergic neurons, we investigated changes in the number and affinity binding of alpha 2-adrenergic receptors on platelets from endurance athletes and a sedentary population using the radiolabeled ligand yohimbine. We found, compared to a control, sedentary population, trained athletes had a 45% increase in the number of platelet alpha 2-adrenergic receptors (338 +/- 39 receptors/platelet vs 233 +/- 25 receptors/platelet, P less than 0.05) with no change in the dissociation constant (2.53 nM +/- 0.2 vs 2.24 nM +/- 0.2, NS) or the ED50 concentration for the competitive displacement of 2.5 nM yohimbine by epinephrine (2.8 X 10(-6) M vs 3.34 X 10(-6) M, NS). The increase in alpha 2-adrenergic receptors found in athletes may explain their decreased catecholamine response and concomitant physiologic responses to exertion.

The endothelium and cyclic guanosine monophosphate in hyperthyroid-induced hypertension.

We investigated the role of endothelium derived relaxing factor (EDRF) and cyclic guanosine monophosphate (cGMP) in the altered vascular reactivity of hyperthyroidism (HT). Rats were given daily injections of triiodothyronine (T3), 50 micrograms/100 g body weight for two weeks, and they had significantly higher serum levels of T3 compared to untreated, control rats (493 +/- 82 vs. 58 +/- 7 ng/dl, p less than 0.05) and significant elevations in their systolic blood pressure (188 +/- 6 vs 126 +/- 3 mm Hg, p less than 0.05). Vascular reactivity was studied in isolated muscle baths; cGMP was measured by RIA. There were no differences in contractile responses to phenylephrine (PE) in isolated aortae from the HT and control rats, but aortae from the HT rats contracted with PE relaxed less to acetylcholine (Ach); the calcium ionophore, A23187; and sodium nitroprusside (SNP). Sensitivity to atrial natriuretic factor (ANF) and 8-Br cGMP was unaltered. Blood vessels from HT rats generated significantly less cGMP in response to Ach, SNP, and ANF. Treatment of the hypertension in the HT rats which hydralazine or propranolol restored the vascular relaxation response to Ach but not SNP; cGMP responses remained blunted. These data suggest that endothelium dependent vasodilators may induce relaxation independent of elevations of cGMP in aortae from HT rats.

The loss of endothelium-dependent vascular relaxation in hypertension.

We investigated endothelium-dependent relaxation in rat aortae, using three models of experimental hypertension: deoxycorticosterone and salt; one-kidney, one clip renovascular hypertension; and coarctation. Isolated aortae were contracted with phenylephrine, and relaxation was subsequently induced with acetylcholine or calcium ionophore A23187. Blood vessels denuded of endothelium did not relax in response to acetylcholine or A23187. Blood vessels from animals with high blood pressure had decreased relaxation responses to acetylcholine and A23187, and also to the endothelium-independent vasodilator sodium nitroprusside. Unlike acetylcholine and A23187, however, nitroprusside completely relaxed the blood vessels from the hypertensive animals, though the sensitivity to nitroprusside was much lower in these vessels. Subsequent reversal of hypertension caused a return of endothelium-dependent relaxation. Loss of endothelium-dependent relaxation occurs readily in the aortae with the development of hypertension; this phenomenon appears to be related to elevated pressure.