Effects of cardiac sympathetic innervation on coronary blood flow.

BACKGROUND: The role of cardiac sympathetic nerves in regulating coronary blood flow is controversial. We sought to determine the degree to which cardiac efferent sympathetic signals modulate coronary blood flow. The heterogeneous sympathetic reinnervation in transplanted hearts provides a model for studying the vasomotor responses to adrenergic stimulation in reinnervated and denervated coronary territories of the same heart. METHODS: We studied 14 cardiac-transplant recipients who had normal coronary arteries and no evidence of rejection and 8 normal subjects. We used positron-emission tomography with [(11)C]hydroxyephedrine, an analogue of norepinephrine, to delineate sympathetic innervation. Using [(13)N]ammonia, we measured myocardial blood flow at rest, during adenosine-induced hyperemia, and in response to sympathetic stimulation induced by cold pressor testing. RESULTS: In the transplant recipients, the uptake of [(11)C]hydroxyephedrine was greater in the territory served by the left anterior descending artery (0.15+/-0.01) than in those served by the right coronary artery (0.07+/-0.01, P<0.001) or the circumflex artery (0.09+/-0.01, P<0.001). The basal flow was similar in all three regions, as was the percent increase in flow during hyperemia. However, the increase in flow in response to cold pressor testing was higher in the territory of the left anterior descending artery (46+/-10 percent) than in those of the right coronary artery (16+/-5 percent, P=0.01) or the circumflex artery (23+/-6 percent, P=0.06), although the changes in hemodynamics and levels of circulating catecholamines were similar. No such regional differences were observed in the normal subjects. CONCLUSIONS: Increases in coronary blood flow in response to sympathetic stimulation correlated with the regional norepinephrine content in the cardiac sympathetic-nerve terminals. These findings suggest that cardiac adrenergic signals play an important part in regulating myocardial blood flow.

Acute reversibility of pulmonary hypertension predicts neither long-term hemodynamic response nor outcome in patients awaiting heart transplantation.

For transplant wait-list patients with end-stage congestive heart failure, reversibility of pulmonary hypertension tested with acute administration of vasodilators is a prerequisite to listing for transplantation. We have shown that the magnitude of the initial pulmonary vasodilatory response to nitroprusside predicts neither the extent of the long-term hemodynamic response nor the subsequent need for transplantation versus clinical improvement and removal from transplant consideration.

Alterations of iodine-131 MIBG biodistribution in an anephric patient: comparison to normal and impaired renal function.

Iodine-131 metaiodobenzylguanidine (MIBG) is an effective agent for the scintigraphic portrayal of pheochromocytomas of all types. Iodine-131 MIBG is a relatively stable radiopharmaceutical that is primarily excreted in the urine. Therefore, impaired renal function would be expected to alter [131I]MIBG pharmacokinetics which would thus affect blood levels, as well as scintigraphy. An 18-yr-old anephric male presented with hypertension and suspected pheochromocytoma. We have compared the [131I]MIBG scintigraphy and blood clearance kinetics in this anephric patient, two patients with renal insufficiency and four patients with normal renal function. The degree of renal insufficiency was directly correlated to the CPM/image (an index of whole-body retention) on all 3 days of imaging and the slower clearance of radioactivity from the blood. The relative distribution of radioactivity between the plasma and cell fractions was greatest in the patients with renal insufficiency. We therefore suggest that attention be paid to plasma creatinine levels prior to the administration of [131I] MIBG to permit accurate interpretation of scintigraphy. In addition, the effect of renal insufficiency on radiation dosimetry should be considered. It may thus be prudent to reduce the administered dose of [131I]MIBG given to anephric or renally insufficient patients to decrease radiation dose.

Metaiodobenzylguanidine as an index of the adrenergic nervous system integrity and function.

The radiopharmaceutical, metaiodobenzylguanidine (MIBG) acts as an analog of norepinephrine (NE). Experiments in rats were carried out to determine how closely the movements of [125I]MIBG in the heart mimicked those of [3H]NE, and if the changes [125I] MIBG concentrations would reflect injury to, and function of, adrenergic neurons in the heart. Injury to adrenergic neurons by 6-hydroxydopamine substantially reduced the uptake of [125I] MIBG into the left ventricle, but the effect was less than that on uptake of [3H]NE uptake and concentration of endogenous NE. Similarly, when desmethylimipramine was given to inhibit the uptake-1 pathway of neurons, the reduction in uptake of [125I]MIBG was statistically significant but less than that of [3H]NE; part of this difference may be attributable to partial uptake of [125I]MIBG into neurons by a diffusion pathway. Substantial fractions of [125I]MIBG and [3H]NE were displaced from the heart by the sympathomimetic drug, phenylpropanolamine. When adrenergic neurons of the heart were stimulated by feeding of rats, the disappearance rates of [3H]NE and [125I]MIBG from the heart were significantly increased. Although not a perfect analog of [3H]NE, [125I]MIBG appears to enter and leave the heart in patterns similar to those of [3H]NE. Thus, movements of [125I]MIBG give indices of adrenergic neuron injury and function in the heart.

Sodium dependency of uptake of norepinephrine and m-iodobenzylguanidine into cultured human pheochromocytoma cells: evidence for uptake-one.

Radioiodinated m-iodobenzylguanidine (MIBG), a scintigraphic agent used in the detection of human pheochromocytomas, is thought to utilize the same uptake and retention mechanism(s) as norepinephrine (NE). Using primary cultures from 16 human pheochromocytomas, we compared the uptake of MIBG to that of NE. Two different uptake systems were identified. Both NE and MIBG were taken up by a sodium-dependent system that was characterized by: temperature dependency, high affinity, low capacity, saturability, ouabain sensitivity, and desmethylimipramine sensitivity. However, NE and MIBG were also taken up by a temperature-dependent, sodium-independent, apparently unsaturable system. The sodium-dependent uptake system fulfills many of the criteria for Uptake-one while the sodium-independent uptake system is most likely a passive diffusion process. Competitive inhibition studies demonstrated that NE and MIBG share a common uptake system; a concept consistent with the linear correlation between the rate of uptake of 1.0 microM NE and that of 1.0 microM MIBG (r = 0.942). At low concentrations, both NE and MIBG entered the tumor cells primarily by the sodium-dependent uptake system. Differential expression of the sodium-dependent and sodium-independent uptake systems, between different tumor cells, appears to be responsible for the variations of the kinetic parameters for both NE and MIBG. These studies provide the first direct characterization of a NE uptake mechanism in human pheochromocytoma cells.

Morphologic and biochemical variability of tissue and cultured cells from human pheochromocytoma.

Primary cell cultures from 18 human pheochromocytomas were maintained in culture for 10 to 12 days and characterized. The cell yields ranged from 1.0 to 60.1 X 10(6) cells/g wet weight of tissue. Cell size, as determined by histofluorescent microscopy, varied as much as seven-fold among cells derived from a given tumor and ten-fold between cells from all tumors. Cell catecholamine content, norepinephrine (NE) plus epinephrine, ranged from 0.4 to 89.5 nmol/10(6) cells at day 5 in culture and did not correlate with catecholamine content of the tissue from which the cells were obtained. Cell catecholamine content decreased with time in culture, but this decrease could not be related to a change in cell viability, the type of media used, an inability to convert dopamine to NE, or an alteration in the uptake of 3H-NE. Cellular uptake of 1.0 microM 3H-NE varied as much as 230-fold between all cell dispersions. The basal and acetylcholine stimulated release of both preloaded 3H-NE and the endogenous catecholamines was quite variable. There was no correlation between the release rate, either basal or stimulated, of preloaded 3H-NE and the endogenous catecholamines. This study represents the largest existing data base on culturing cells from these tumors and describes many of the morphologic and biochemical characteristics of this cell system.

Iodine-123-4-amino-3-iodobenzylguanidine, a new sympathoadrenal imaging agent: comparison with iodine-123 metaiodobenzylguanidine.

Iodine-123-4-amino-3-iodobenzylguanidine ([123I]AIBG), an analog of 123I metaiodobenzylguanidine ([123I]MIBG), has an advantage in having a more rapid and simple synthesis. This, combined with animal data that suggested a greater affinity of the new radiopharmaceutical for the autonomic innervation of the myocardium led us to study the biodistribution of [123I]AIBG in three men with metastatic pheochromocytoma. In all instances, [123I]AIBG revealed the same metastatic deposits shown by [123I]MIBG. Iodine-123 AIBG uptake, however, was greater than [123I]MIBG in lung, gut, and spleen. These higher backgrounds may pose diagnostic problems in some cases.

Metabolism of iodine-131 metaiodobenzylguanidine in patients with metastatic pheochromocytoma.

Iodine-131 metaiodobenzylguanidine ([131I]MIBG) is used to image and treat human pheochromocytoma. As part of a pharmacodynamic study of this agent, we have evaluated its excretion and metabolism in nine pheochromocytoma patients undergoing MIGB therapy. Following diagnostic doses of [131I]MIBG given prior to therapy, 40 to 55% of the administered radioactivity generally appeared in the urine within 24 hr and 70 to 90% was recovered within 4 days. Reverse-phase high performance liquid chromatography was used to identify radioactive metabolites following therapeutic doses of [131I]MIBG. Unaltered [131I]MIBG was the major radioactive urinary component found, representing 75 to 90% of the total in all but one of the nine patients examined. The urine samples from the patient, whose rate of urinary excretion was the lowest of the group, contained [131I]-m-iodohippuric acid ([131I]MIHA) in amounts equal to that of [131I]MIBG, as well as small amounts of [131I]iodide and [131I]-m-iodobenzoic acid ([131I]MIBA). Iodine-131 MIHA and [131I]iodide were also minor components in the urine samples from the other eight patients. Trace quantities of [131I]MIBA and 131I-4-hydroxy-3-iodobenzylguanidine ([131I]HIBG) were also detected in a few of the patient urine samples examined. The 4- to 5-day metabolism profiles varied from patient to patient but were similar for the same patient following therapy doses given 4 mo apart. There was no obvious correlation between the presence of metabolites and the location of the tumors or the plasma or urinary catecholamine levels. Extraction of radioactivity from two pheochromocytomas removed from patients was determined to be primarily MIBG. These studies suggest that [131I]MIBG is a rapidly excreted, relatively stable radiopharmaceutical agent.

Rapid analysis for methylglyoxal bis(guanylhydrazone) by reversed-phase ion-pair liquid chromatography.

We describe a "high-performance" reversed-phase ion-pair liquid-chromatographic procedure for measuring methylglyoxal bis(guanylhydrazone) (MGBG) in plasma, urine, and bone-marrow leukocytes. Specimens of plasma and bone-marrow leukocytes are deproteinized with perchloric acid, then neutralized with KOH. Urinary MGBG is isolated by liquid-solid extraction in a C18 Sep-Pak. The chromatographic system consists of a 45 X 4.6 mm (i.d.) octadecylsilyl (C18, 5-microns particle) column and a mobile phase consisting of methanol/sodium acetate buffer (200 mmol/L, pH 4.5), 2/3, by vol. The acetate buffer also contains 20 mmol of 1-octanesulfonate and 40 mg of sodium azide per liter. The column effluent is monitored at 283 nm. At a flow rate of 3.0 mL/min, MGBG is eluted in 1.67 min. The detection limit is 20 nmol/L, and peak height varies linearly with concentration from 0.02 to 40 mumol/L. Analytical recovery exceeds 99%. Within-day CVs ranged from 0.9% to 2.9%, between-day CVs from 4.2% to 6.2%.

Effect of uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine.

The mechanisms underlying the uptake of the radiopharmaceutical metaiodobenzylguanidine (MIBG) and the catecholamine norepinephrine (NE) were studied using cultured bovine adrenomedullary cells as an in vitro model system. Sodium-dependent and sodium-independent uptake systems have been identified and characterized for both MIBG and NE. The sodium-dependent uptake of NE and MIBG was inhibited by the selective Uptake-one inhibitors, desmethylimipramine (DMI) and cocaine, whereas the sodium-independent uptake for NE and MIBG was much less sensitive to inhibition by these agents. The sodium-dependent uptake system fulfills the criteria for the neuronal Uptake-one system, and the sodium-independent uptake system fulfills the criteria for a passive diffusion mechanism. Both NE and MIBG were transported into cultured bovine adrenomedullary cells by both uptake systems; the relative role of each uptake system was dependent upon the concentration of NE and MIBG in the media. Arterial concentrations proximal to the dog adrenal were very small suggesting that the sodium-dependent (Uptake-one) system is predominant in vivo. Consistent with the in vitro observations, the in vivo uptake of MIBG and NE into dog adrenal medullae was effectively blocked by pretreatment with DMI or cocaine. Therefore, iodine-131 MIBG scintigraphy of the adrenal appears to reflect uptake by way of the Uptake-one system.

Adrenal cortical 11 beta-hydroxylase and side-chain cleavage enzymes. Requirement for the A- or B-pyridyl ring in metyrapone for inhibition.

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.

Comparison of the sodium dependency of uptake of meta-lodobenzylguanidine and norepinephrine into cultured bovine adrenomedullary cells.

Radioiodinated meta-iodobenzylguanidine (MIBG), a scintigraphic agent used for the detection of human pheochromocytomas, is thought to utilize the same uptake and retention mechanism(s) as norepinephrine (NE). Using cultured bovine adrenomedullary cells, we compared the mechanism(s) of uptake of MIBG to that of NE. Two different uptake systems were identified. NE and MIBG were taken up by a sodium-dependent system that was characterized by: 1) temperature dependency; 2) high affinity: Km of 1.22 +/- 0.12 microM for MIBG and 1.41 +/- 0.50 microM for NE; 3) low capacity: Vm (picomoles/10(6) cells/10 min) of 64.3 +/- 3.3 for MIBG and 36.6 +/- 7.2 for NE; 4) saturability; 5) ouabain sensitivity; and 6) energy dependency. However, NE and MIBG also were taken up by a temperature-dependent, sodium-independent, apparently unsaturable, and energy-independent system. The sodium-dependent uptake system fulfills many of the criteria for Uptake1 whereas the sodium-independent uptake system is most likely a passive diffusion process. NE uptake proceeded predominantly by the sodium-dependent process. Uptake of MIBG occurred by both pathways at low concentrations, but at high concentrations (greater than 10 microM) uptake was predominantly (75 to 100%) by the sodium-independent process. Inhibition studies suggest that MIBG and NE are transported by the same carrier involved in the sodium-dependent system. Scintiscans of the human adrenals and pheochromocytomas appear to reflect uptake of [131I]MIBG by the sodium-dependent system.

Structure-activity relationship study of the inhibition of adrenal cortical 11 beta-hydroxylase by new metyrapone analogues.

Metyrapone, 2-methyl-1,2-di-3-pyridyl-1-propanone (1a), is a potent reversible inhibitor of the cytochrome P-450 11 beta-hydroxylase enzyme system (P-450(11) beta) of the adrenal cortex. The structural features of the metyrapone molecule have been systemically altered to determine the requirements necessary for inhibition of P-450(11) beta activity. Metyrapone and 14 analogues have been obtained or synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. The inhibition of P-450(11) beta activity with these derivatives demonstrated that (1) the A-ring phenyl derivatives 2a-d were better inhibitors than the respective dipyridyl analogues, (2) the ketone in the 1-position can be replaced by various functionalities without markedly reducing inhibition, and (3) at least one methyl group should be present in the 2-position to maintain inhibition. The observed inhibition of P450(11) beta activity with the metyrapone analogues suggest that A-ring phenyl metyrapone analogues 2a-d would be candidates for radioiodination and subsequently used as adrenal cortical imaging agents.

Absorption of 4,4'-methylenebis [2-chloroaniline] by human skin.

A system was developed to measure percutaneous absorption of water-insoluble environmental agents into human skin. Percutaneous absorption of 4,4'-[14C]methylenebis [2-chloroaniline] (MBOCA) was measured during dry contact exposure of MBOCA with organ cultures of neonatal foreskin. Time-dependent exposures, autoradiographs, and thin-layer chromatography indicated that MBOCA was rapidly and progressively absorbed and passed through the skin without being metabolized. The transepithelial penetration of MBOCA was temperature dependent. Under certain conditions, the stratum corneum contained more MBOCA than other layers in the skin, which suggested that the stratum corneum may not be the main barrier for percutaneous absorption of MBOCA. An assessment of risk confirmed that skin exposure is a cause for concern.

Kinetics of tissue distribution and elimination of 4,4'-methylene bis(2-chloroaniline) in rats.

The tissue distribution kinetics and elimination of 4,4'-methylene bis(2-chloroaniline) (MBOCA) in rats was studied after a single dose of [14C]MBOCA (0.49 mg/kg body weight, i.v.). The highest concentrations of radioactivity were in the small intestine, liver, adipose, lung, kidney, skin, and adrenals. For most tissues, a rapid decrease in radioactivity was followed by a slower decrease except for the small intestine, adipose and skin which demonstrated transient increases. Subcellular distribution in liver at 1 h showed radioactivity in all cell fractions. Although very lipophilic, [14C]MBOCA was completely eliminated within 48 h with the major route via the feces (73.4%).

Scintigraphic localization of pheochromocytoma.

We used a new radiopharmaceutical agent, [131I]meta-iodobenzylguanidine ([131I]MIBG), to produce scintigraphic images of pheochromocytomas in eight patients. One day or more after injection, the only normal organ that displayed distinct concentrations of radioactivity was the urinary bladder. The [131I]MIBG was probably concentrated in adrenergic vesicles; in tissues where vesicles are numerous, such as pheochromocytomas, the radionuclide was retained for days. The spectrum of pheochromocytomas shown the scintigrams was broad: intra-adrenal and extraadrenal in location, benign and malignant in character, 0.2 to 65 g in weight, and with different hormone patterns in secretion. Tumors in four patients were not detected by computed tomography. In one patient, reoperation was undertaken only because the scintigram located the extra-adrenal tumors and thereby directed the surgeon's exploration. The method offers hope of safe and reliable localization of pheochromocytomas in their many guises.

Imaging the primate adrenal medulla with [123I] and [131I] meta-iodobenzylguanidine: concise communication.

An evaluation of radioiodinated meta-iodobenzylguanidine (m-IBG) as an adrenomedullary imaging agent is reported in 15 rhesus monkeys. Scintiscans of the monkey adrenal medulla have been obtained with [123I]- and [m-131I]IBG at 2-6 days after injection. The imaging superiority of m-IBG over its positional isomer, para-iodobenzylguanidine (p-IBG), is documented in both dogs and monkeys. Administration of reserpine, a depletor of catecholamine stores, markedly lowers the [m-131I]-IBG content of the dog adrenal medulla, but the adrenergic blocking agents phenoxybenzamine and propranolol have no effect. Subcellular fractionation of the dog's adrenal medullae reveals that m-IBG is sequestered mainly in the chromaffin storage granules. The results of this study suggest that radioiodinated m-IBG, previously reported to image the primate myocardium, also merits evaluation as a clinical radiopharmaceutical for the adrenal medulla.

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase. Purification, characterization, and further evidence for identity.

Alpha-Aminoadipate aminotransferase and kynurenine aminotransferase activities were co-purified from the rat kidney supernatant fraction. The resulting preparation was determined to be nearly homogenous by analytical disc gel electrophoresis at pH 8.9 and 7.5 isoelectric focusing on polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of approximately 85,000 was determined on Sephadex G-200 chromatography and sucrose density gradient analysis. The enzyme was determined to be comprised of two subunits of approximately the same molecular weight (45,500 +/- 850) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An isoelectric pH of 6.56 +/- 0.06 was determined by focusing on polyacrylamide gels. Further evidence is provided to support the idea that the alpha-aminoadipate aminotransferase and kynureine aminotransferase activities are properties of a single protein: (a) co-purification of the two activities from the rat kidney supernatant fraction with the ratio of their specific activities remaining constant, (b) similar chromatographic behavior, (c) a similarity in their dependence on added pyridoxal-P for activity, and (d) a similar pattern of heat inactivation.