O. Bryan Holland

Angiotensin increases aldosterone synthase mRNA levels in human NCI-H295 cells

Understanding the regulation of aldosterone secretion has been hampered by the lack of a cell culture system that remains chronically responsive to angiotensin stimulation. NCI-H295 cells, cultured from a human adrenocortical tumor, express the three major pathways of adrenal steroidogenesis and produce small amounts of aldosterone during basal culture. We have determined changes in aldosterone production and in aldosterone synthase (AS, P45011B2) mRNA levels in these cells in response to angiotensin II (AII) and forskolin. Culture of NCI-H295 cells with 10(-7) M AII or with 10(-5) M forskolin stimulated aldosterone production and increased AS mRNA levels, though the effect of AII was greater. When cells were cultured with increasing concentrations of AII from 10(-11) through 10(-8) M, a dose-dependent increase in AS mRNA levels paralleled increases in aldosterone production. In view of these findings, these human adrenocortical cells should be useful for exploring mechanisms regulating aldosterone production.

Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart

The effects of calcitonin gene-related peptide (CGRP) on heart rate, coronary flow, pressure development, and time to ischemic contracture were studied in the isolated, perfused rat heart. A bolus of CGRP (2640 pmols) caused significant increases in heart rate and coronary flow; these effects were sustained for at least five minutes after injection. The increase in coronary flow was independent of heart rate, since CGRP caused an increase in coronary flow in non-beating (potassium-arrested) hearts. The dose-response of CGRP was studied using five doses (65, 218, 658, 1320 and 2640 pmols) given as bolus injections. Although the increase in heart rate was apparently dose-dependent, significant increases above baseline were observed only with the two highest doses. In contrast, coronary flow increased significantly above baseline with the injection of all but the lowest dose of CGRP. Ten minutes after injection of CGRP, all hearts were made ischemic. The time to onset of ischemic contracture was approximately 11 minutes for those hearts that received 65 pmols of CGRP; however, for those hearts receiving all other doses of CGRP, the time to onset of contracture was approximately 8 minutes. We conclude that CGRP significantly decreases the resistance of the coronary vascular bed, and that it may be an important regulator of regional blood flow in the heart.

Decreased spinal cord content of calcitonin gene-related peptide in the spontaneously hypertensive rat

Calcitonin gene-related peptide (CGRP), produced by alternative processing of the primary transcript of the calcitonin gene, is a potent vasodilator. We have shown that dietary calcium deficiency accompanied by decreased serum ionized calcium significantly decreases the neuronal content of CGRP in laminae I and II of the dorsal horn of the spinal cord in the growing rat. The spontaneously hypertensive rat (SHR) is characterized by decreased serum ionized calcium levels and is thought to most closely resemble human essential hypertension. To determine if the neuronal content of CGRP is decreased in the SHR compared to the Wistar-Kyoto (WKY) parent strain, CGRP was localized immunocytochemically in the dorsal horn of the spinal cord. The density of immunocytochemical staining was quantitated by computer-assisted image processing of laminae I and II of the upper thoracic spinal cord of 12-14 week old male SHR (n = 4) and WKY (n = 4) normotensive, control rats. The SHR had significantly decreased neuronal CGRP content compared to the WKY rats (107 +/- 5 vs 121 +/- 6 arbitrary units, P less than 0.01). In contrast, the neuronal density of substance P (SP), which frequently co-exists with CGRP in this neuronal population, was not different between the two groups (SHR, 91 +/- 6 (n = 4) vs WKY, 88 +/- 3 arbitrary units (n = 4)).

Dietary calcium modulates spinal cord content of calcitonin gene-related peptide in the rat.

This study was undertaken to determine if calcium status modulates calcitonin gene-related peptide (CGRP) neuronal content. In two separate experiments, young, growing rats and mature rats were placed on low, normal, and high calcium diets for four weeks. CGRP immunostaining was localized immunocytochemically in laminae I and II of the upper thoracic spinal cord in young rats and in the upper thoracic and lumbar spinal cord in mature rats. Low calcium intake decreased dorsal horn CGRP content in young, growing rats, while high calcium diet significantly increased CGRP content, as determined by computer-assisted image processing, in both young and adult rats. A significant positive correlation was found between the serum ionized calcium and CGRP content in laminae I and II. Thus, calcium balance appears to modulate neuronal CGRP content.

Potassium Loss, Ventricular Irritability, and the Risk of Sudden Death in Hypertensive Patients

SummaryIn the past, potassium depletion in both non-digitalised patients and in patients without cardiac disease was thought to cause no adverse cardiac effects. However, several studies have now demonstrated a significant incidence of ventricular ectopic activity (VEA) with diuretic-induced hypokalaemia, even in hypertensive patients without overt heart disease. Additional evidence suggests that sudden death may occasionally result from this VEA. Potassium repletion with potassium-sparing diuretics or with potassium chloride supplementation has generally demonstrated a beneficial therapeutic effect in reducing VEA. However, after diuretic therapy occasional patients may have persistent VEA which may result from focal myocardial lesions associated with potassium depletion. In contrast, diuretic therapy in which normokalaemia is maintained has only been associated with a very low occurrence of VEA. Thus, with the preservation of normokalaemia, diuretic therapy for hypertension does not appear to be associated with the significant hazards of VEA.RésuméAuparavant, une déplétion en potassium chez des malades non digitalisés et chez des malades indemnes de maladie cardiaque était censée n’avoir aucun effet cardiaque nocif Cependant, plusieurs études ont maintenant montré qu’il existe une incidence significative d’activité ectopique ventriculaire (A.E.V.) avec les diurétiques inducteurs d’hypokaliémie même chez les hypertendus sans maladie cardiaque patente. Des preuves s’ajoutent pour suggérer que cette A.E. V. peut éventuellement conduire à une mort subite. Une réplétion en potassium avec des diurétiques d’épargne du potassium ou avec un supplément en chlorure de potassium s’est généralement montrée d’un bénéfice thérapeutique intéressant en réduisant cette A.E.V. Cependant, après un traitement diurétique, certains malades sont susceptibles d’avoir uneA.E. V. persistante qui peut résulter de lésions myocardiques focalisées associées à la déplétion potassique. A l’inverse, le traitement diurétique pendant lequel on maintient une normokaliémie n’a été associé qu’à une très faible survenue d’A.E. V. Ainsi, en préservant une normokaliémie, le traitement diurétique à visée antihypertensive n’apparait pas associé de façon significative à des problèmes d’A.E. V.ZusammenfassungIn der Vergangenheit glaubte man, daβ ein Kalium-Mangel sowohl in nicht-digitalisierten Patienten als auch in Patienten ohne Herzkrankheit keine kardialen Nebenwirkungen erzeugt. In verschiedenen Studien wurde jedoch jetzt eine signifikante Häufigkeit einer ventrikulären ektopischen Aktivität (VEA) mit der Diuretika-induzierten Hypokaliämie selbst bei Hypertonikern ohne offensichtliche Herzkrankheit nachgewiesen. Zusätzliche Hinweise lassen vermuten, daβ gelegentlich ein plötzlicher Herztod Ergebnis dieser VEA sein kann. Eine Kalium-Auffüllung mit Kalium-sparenden Diuretika oder mit Supplementen von Kaliumchlorid zeigte im allgemeinen einen günstigen therapeutischen Effekt bei der Reduktion der VEA. Nach einer diuretischen Therapie können jedoch gelegentlich bei Patienten persistierende VEA als Ergebnis von mit einem Kalium-Mangel im Zusammenhang stehenden fokalen myokardialen Läsionen vorliegen. Dagegen kommt bei einer diuretischen Therapie, in der eine Normokaliämie aufrecht erhalten wird, es nur zu einem sehr niedrigen Auftreten einer VEA. Bei Bewahrung einer Normokaliämie scheint daher die diuretische Therapie bei der Hypertonie nicht mit den bedeutenden Gefahren einer VEA in Verbindung zu stehen.ResumenAntaño se creía que el agotamiento de las reservas de potasio no causaba efectos cardiacos adversos en los pacientes no digitalizados y en los que no padecían enfermedad cardiaca; sin embargo, varios estudios han demostrado que hay una incidencia significativa de actividad ventricular extópica (AVE) incluso en los pacientes hipertensos sin cardiopatía manifiesta. Otros datos adicionales sugieren que la muerte repentina puede derivarse ocasionalmente de esta AVE. La repleción potasica con diuréticos ahorradores de potasio o con suplementos de cloruro potásico ha tenido por lo general un efecto terapéutico beneficioso reduciendo la A VE; sin embargo, después de la terapéutica diurética algunos pacientes muestran una A VE persistente que resultaría de lesiones miocárdicas focales asociadas al agotamiento de potasio. En cambio, la terapéutica diurética en la que se mantiene la normocaliemia sólo ha aparecido asociada a una baja incidencia de AVE. Por tanto, preservando la normocaliemia, la terapéutica diurética de la hipertensión no parece ir acompañada de los riesgos de la AVE.ResumoNo passado, considerava-se que a depleção de potássio tanto em pacientes rão digitalizados como nos pacientes sem doença cardíaca, não causasse efeitos cardíacos adversos. Entretanto, diversos estudos revelaram uma incidência significante de atividade ectópica do ventrículo (AEV), acompanhada de hipopotassemia induzida por diuréticos, mesmo em pacientes com hipertensão mas sem doença cardíaca manifesta. Outros dados evidenciam que a morte súbita pode resultar ocasionalmente de uma tal AEV. A reposição de potássio por meio de diuréticos poupadores de potássio ou de suplementação com cloreto de potássio geralmente demonstrou ser eficaz para a redução de AEVs. Contudo, após terapia com diuréticos, pode ocorrer que as AEVs persistam em alguns pacientes, possiveimente como resultado de lesões focais do miocárdio associadas à depleção de potássio. Por outro lado, a terapia com diuréticos, em que se mantém a concentração de potássio, foi associada a apenas uma baixa ocorrência de AEVs. Desse modo, em se preservando as concentrações normais de potássio, o tratamento com diuréticos para a hipertensão não parece estar associado a riscos significativos de AEVs.RiassuntoNegli anni scorsi si riteneva che la deplezione potassica non causasse effetti cardiaci dannosi in pazienti non digitalizzati o senza cardiopatie. Tuttavia parecchi studi hanno ora dimostrato una significativa incidenza di ectopie ventricolari (EV) in corso di ipopotassiemia indotta da diuretici, anche in pazienti ipertesi senza una cardiopatia manifesta. Ulteriori evidenze suggeriscono che la morte improvvisa possa svilupparsi a seguito di queste EV. L’uso di diuretici risparmiatori di potâssio o supplement di cloruro di potassio si sono in genere dimostrati utili nel ridurre le EV. Tuttavia, dopo la terapia diuretica alcuni pazienti possono avere persistenti EV che possono essere causate da lesioni miocardiche focali associate a deplezione potassica. Al contrario, in corso di trattamento diuretico associato a normale potassiemia sono state osservate molto raramente EV. Pertanto, mantenendo normali livelli plasmatici di potassio, la terapia dell’ipertensione con diuretici non sembra associata a rischi significativi di EV.

Role of 18-hydroxylated cortisols in hypertension

The isolation of 18-hydroxycortisol and 18-oxocortisol was recently described. These steroids have been shown to be excreted in exaggerated quantities in patients with primary aldosteronism, with adrenal adenomas and in glucocorticoid suppressible aldosteronism. We report the measurement of both steroids in the urine of patients with essential hypertension. 18-Oxocortisol excretion did not differ in patients with normal renin essential hypertension (0.7 +/- 0.7 micrograms/24 h), low renin essential hypertension (0.7 +/- 0.5 micrograms/24 h) and normal individuals (1.2 +/- 0.9 micrograms/24 h). Patients with normal renin hypertension excreted 54 +/- 43 micrograms/24 h of 18-hydroxycortisol, those with low renin essential hypertension excreted 58 +/- 54 micrograms/24 h, and normal individuals excreted 63 +/- 36 micrograms/24 h. Three of the low renin and one of the normal renin hypertensive subjects excreted greater quantities of 18-hydroxycortisol than the upper limit of normal, but all excreted normal quantities of 18-oxocortisol. As 18-hydroxycortisol is inactive, the meaning of this elevated excretion is unclear, but it may be a marker of an adrenal enzymatic abnormality which may be playing a more direct role in hypertension.

Aldosterone synthase gene regulation by angiotensin.

Excessive aldosterone secretion in some hypertensive patients may result from abnormal aldosterone synthase (AS) gene regulation in response to changes in dietary sodium intake. We have utilized NCI-H295 cells, which exhibit stable angiotensin-induced aldosterone secretion, for transient transfections with murine AS/human growth hormone reporter constructs. An angiotensin response element increasing AS gene transcription during angiotensin stimulation appears to reside within the initial 425 nt of the murine AS promoter. We also noted the possible presence of a negatively-acting cis element between nt -425 and -1500. These studies provide an initial step toward characterizing molecular mechanisms by which angiotensin regulates AS gene transcription.

Metabolic changes with antihypertensive therapy of the salt-sensitive patient

Recent evidence suggests that metabolic changes that occur with antihypertensive agents may influence cardiovascular risk. Diuretic therapy is particularly appropriate for the salt-sensitive hypertensive patient. However, diuretic-induced electrolyte abnormalities may lead to ventricular arrhythmias, even in patients with uncomplicated essential hypertension. Antihypertensive drugs may change circulating lipoprotein levels, which may influence the development of atherosclerosis. Therefore, serum cholesterol and triglyceride levels should be monitored when antihypertensive drugs are administered that can cause hyperlipidemia. Weight reduction and diet therapy should be used because these may have a greater effect on reducing hyperlipidemia, though choice of antihypertensive agents is important. In addition, glucose tolerance may worsen with thiazide therapy, perhaps because newer evidence suggests that insulin resistance is common in essential hypertension. This glucose intolerance may be corrected with potassium repletion or substitution of bumetanide for thiazide. The calcium antagonists may be substituted for diuretic therapy, or other classes of antihypertensive drugs may be used with a reduced dose of diuretic drug if these metabolic changes persist. Thus, attention to metabolic changes may be as important as blood pressure reduction in treatment of the salt-sensitive hypertensive patient.

18-Oxocortisol: Effect of dexamethasone, ACTH and sodium restriction

The urinary excretion of 18-oxocortisol in 37 normal subjects consuming a normal sodium diet was 1.2 +/- 0.9(SD) microgram/24 h. Dexamethasone administration to 5 normal individuals suppressed the excretion of 18-oxocortisol from 1.16 +/- 0.5 micrograms/24 h to 0.6 +/- 0.2 micrograms/24 h. While they still received dexamethasone, ACTH administration raised the 18-oxo-cortisol excretion to 3.82 +/- 1.2 micrograms/24 h. Seven normal subjects were placed on a sodium restricted diet, and the urinary excretion of 18-oxocortisol rose from 1.5 +/- 1.21 micrograms/24 h to 8.54 +/- 5.08 micrograms/24 h and aldosterone from 6.6 +/- 2.0 micrograms/24 h to 39.7 +/- 14.6 micrograms/24 h. Two of the seven individuals showed minimal increases in the excretion of 18-oxocortisol, but in all cases aldosterone increased with sodium restriction. The urinary excretion of 18-oxocortisol correlated significantly with the excretion of aldosterone, 18-hydroxycortisol, cortisol, and 19-nordeoxycorticosterone. These studies indicate that 18-oxocortisol secretion is under ACTH regulation, but since sodium restriction also increases the excretion of 18-oxocortisol, the renin-angiotensin system must also participate in its regulation. However, some individuals do not increase their excretion of 18-oxocortisol with sodium restriction, although aldosterone excretion increases as expected, suggesting that additional factors participate in the regulation of 18-oxocortisol production.

Drug-induced hypokalaemia. A cause for concern.

Hypokalaemia (defined as a plasma potassium concentration<3.5 mEq/L) is a common electrolyte abnormality in clinical practice. Drugs are a common cause of either asymptomatic or symptomatic hypokalaemia. Drug-induced hypokalaemia is an important problem particularly in the elderly and in patients with cardiovascular, renal or hepatic disease. Hypokalaemia can complicate the use of the drug in the therapeutic concentration range, and can also be precipitated with overdose or conditions leading to drug intoxication. Because the etiologies of hypokalaemia are numerous, the diagnosis of drug-induced hypokalaemia may be overlooked. Physicians should always pay close attention to this common side effect. Evaluation and management of a hypokalaemic patient should include a careful review of medications history to determine if a drug capable of causing or aggravating this electrolyte abnormality is present.SummaryDrug-induced hypokalaemia is a widespread problem in the elderly that can be caused by many therapeutically useful substances, the most common of which are diuretics. In certain classes of patients (e.g. those with acute myocardial infarction, with congestive heart failure receiving digitalis, or with cirrhosis), iatrogenic hypokalaemia is an established risk factor. In patients with hypertension who have no underlying heart disease or liver disease, the use of diuretics may lead to worsened glucose tolerance and cardiac arrythmias. There is also evidence for an increased risk of sudden cardiac death.