Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome.

BACKGROUND AND PURPOSE: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. EXPERIMENTAL APPROACH: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. KEY RESULTS: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.

Successful treatment of malignant neurocardiogenic syncope with repeated tilt training program.

Recent reports have shown that repeated tilt-table testing or tilt training is a very effective therapy for the treatment of neurocardiogenic syncope induced by head-up tilt testing. The present patient experienced repeated syncopal or presyncopal attacks and had shown prolonged asystole on an electrocardiogram during syncope. The presyncope could be reproducibly induced by head-up tilt testing. Oral propranolol and/or disopyramide therapies failed to prevent his symptoms. Tilt training (2 sessions/day) was repeated every day for 4 weeks at home, and then head-up tilt testing was performed again. The syncope or presyncope was not induced by head-up tilt testing. The patient has continued with this training and has had no symptoms during the follow-up period of 1 year.

Heart rate and plasma cyclic AMP responses to isoproterenol infusion and effect of beta-adrenergic blockade in patients with postural orthostatic tachycardia syndrome.

To clarify the pathophysiological mechanism of postural orthostatic tachycardia syndrome (POTS). the responses of heart rate and the plasma cyclic AMP (cAMP) level to isoproterenol infusion in the supine position were evaluated in 10 POTS patients and 10 age-matched controls. Also, the effect of beta-adrenergic blockade on POTS was assessed using the headup tilt test. The POTS patients consisted of two men and eight women, with a mean age of 19.7 years (range. 15-28 years). An exaggerated increase of both heart rate and the plasma cAMP concentration after isoproterenol infusion at a low dose (1 microg/min) was observed in the POTS patients, but not in control subjects. Seven POTS patients received oral beta-blocker therapy. In five of these, symptoms were abolished and there was a smaller heart rate increase during the head-up tilt test. These data suggest that POTS may be more prevalent in young women and that beta-adrenergic hypersensitivity may play a role in the mechanisms of this syndrome. However, a larger series of patients need to be studied in the future.

Treatment of malignant neurocardiogenic vasovagal syncope with a rate drop algorithm in dual chamber cardiac pacing.

A 29-year-old man with malignant vasovagal syncope presented with episodes of abrupt loss of consciousness associated with an aura, totaling more than 10 episodes over 3 months. Holter monitoring showed cardiac arrest with a duration of 15 seconds. Oral propranolol and disopyramide therapy failed to prevent the syncope. A dual chamber pacemaker with a rate drop response algorithm successfully prevented the syncope but not the aura. There may be multifactors involved in the mechanism of this syndrome. The patient has returned to a normal active life. This rate drop algorithm is an effective therapy for the prevention of syncope in malignant vasovagal syncope.

Comparison of binding affinities of omega-conotoxin and amlodipine to N-type Ca2+ channels in rat brain.

AIM: To compare the binding affinities of omega-conotoxin (CTX) and amlodipine to N-type Ca2+ channels in rat brains. METHODS: Whole rat brains were homogenized in HEPES buffer 50 mmol.L-1 (pH 7.4) and centrifuged at 40,000 x g to obtain the membrane-entriched fraction. 125I-omega-conotoxin (125I-omega-CTX) was used as a radioligand. Using radioligand binding assay Kd and Bmax values of the radioligand were determined by Scatchard analysis. The IC50 value for each drug was obtained from displacement experiments. RESULTS: No differences in Bmax values of 125I-omega-CTX binding sites between frozen and fresh tissues were observed. Values of Kd and Bmax of N-type Ca2+ channels were 0.02 +/- 0.01 nmol.L-1 and 1029 +/- 108 pmol/g protein, respectively. The pKi values of omega-CTX and amlodipine were 9.57 and less than 4, respectively. The pKi values of propranolol, prazosin, atropine, and histamine were very low. CONCLUSION: The binding affinity of the L-type Ca(2+)-antagonist amlodipine to N-type Ca2+ channels in the rat brain was very low.

Alpha-1 adrenoceptor subtypes (high, low) in human benign prostatic hypertrophy tissue according to the affinities for prazosin.

BACKGROUND: A novel classification of alpha-1 adrenoceptor subtypes (High, Low) was applied to human benign prostatic hypertrophy (BPH) tissue. METHODS: Human BPH specimens were examined by a radioligand binding assay method using 3H-prazosin, and those data were compared with preoperative therapies. RESULTS: (1) Scatchard analysis showed a high-affinity site (Kd:27.18 +/- 6.41 pM; Bmax:9.29 +/- 0.98 fM/mg protein; mean +/- SE) as alpha 1H, and a low-affinity site (Kd: 4088.0 +/- 744.34 pM, Bmax: 140.81 +/- 19.98 fM/mg protein) as alpha 1L subtype, for prazosin. (2) The Kd and Bmax were not different in the nontreated group (n = 5), alpha 1 blocker group (n = 5), and antiandrogen group (n = 5), in either alpha 1-high affinity or alpha 1-low affinity subtype. (3) Phenoxybenzamine had different pKi values for the above two adrenoceptor subtypes. Scatchard analysis showed that alpha 1-high affinity binding site disappeared in the presence of 1 microM of phenoxybenzamine, and the Kd and Bmax values in the presence of 1 microM of phenoxybenzamine were almost identical to the alpha 1-low affinity site of the two subtypes. CONCLUSIONS: Human BPH tissue possesses both alpha 1H- and alpha 1L-adrenoceptor subtypes according to the affinities for prazosin, and only the alpha 1H subtype can be completely inhibited by some concentration of phenoxybenzamine. Treatment by alpha 1 blocker may not change the conditions of alpha 1-adrenoceptors in prostatic tissue.

Affinity for [3H]iloprost binding sites and cAMP synthesis activity of a 3-oxa-methano prostaglandin I1 analog, SM-10906, in human platelets and endothelial cells.

SM-10902 ((+)-methyl [2-[(2R,3aS,4R,5R,6aS)-octahydro-5-hydroxy-4-[(E)-(3S,5S)-3- hydroxy-5-methyl-1-nonenyl]-2-pentalenyl]ethoxy]acetate) and its free acid, SM-10906 are new stable 3-oxa-methano prostaglandin (PG) I1 analogs. Their affinities for [3H]iloprost and [3H]PGE2 binding sites in human platelets and human umbilical vascular endothelial cells were compared with those of the PGI2 analog iloprost, PGE1 and PGE2 by the radioligand binding assay method. The cyclic AMP (cAMP) synthesis activity of these drugs were also determined in human umbilical vascular endothelial cells. We found that SM-10906 apparently displaced [3H]iloprost binding to the membrane fractions in those cells since the pKi values were 6.30 in platelets, 7.52 in vein endothelial cells and 6.31 in the arterial endothelial cells. The pKi values of SM-10906 for [3H]PGE2 binding sites were significantly lower than those obtained for [3H]iloprost binding. SM-10902, which is a prodrug of SM-10906, showed low affinity for [3H]iloprost binding sites in those cells. SM-10906 also dose-dependently enhanced the cAMP level in the vascular endothelial cells. Thus, these findings indicate that SM-10906 binds to [3H]iloprost binding sites and exhibits pharmacological functions such as an anti-platelet action and a cytoprotective action in endothelial cells through the elevation of intracellular cAMP contents.

Proadrenomedullin N-terminal 20 peptide (PAMP) reduces inward currents and Ca2+ rises induced by nicotine in bovine adrenal medullary cells.

It has been recently reported that proadrenomedullin N-terminal 20 peptide (PAMP), which is secreted with adrenomedullin and catecholamines from the adrenal medulla, inhibits catecholamine release stimulated with nicotine. In the present study, to elucidate anticholinergic mechanisms of PAMP we employed the whole-cell patch-clamp and the intracellular Ca2+ imaging techniques in cultured bovine adrenal medullary cells. PAMP inhibited nicotinic currents and [Ca2+]i rises induced by nicotine in a dose-dependent manner (10(-9)-10(6) M). These inhibitions were selective, since PAMP alone did not induce any ionic currents, moreover it did not affect voltage-dependent Ba2+ currents or high K+ (50 mM)-induced [Ca2+]i rises. The onset of the inhibitory effect of PAMP (10(-6) M) was very rapid and reached a steady-state level within 10 sec. The effect of PAMP (10(-6) M) lasted for about 10-15 min. Desensitization process of the nicotinic current fitted to a single exponential function with a time constant of 6.4 +/- 0.3 sec. When PAMP (10(-6) M) simultaneously added with nicotine (10(-5) M), the desensitization process was facilitated and fitted to two exponentials with time constants of 0.46 +/- 0.08 and 2.5 +/- 0.8 sec. From the present results, the inhibition by PAMP of nicotinic currents which was well associated with that of nicotine induced [Ca2+]i rises leads to the attenuation of catecholamine release probably, at least in part, due to the facilitation of the desensitization process of the nicotinic currents.

Immune responsiveness in Mycobacterium avium-infected mice: changes in the proportion of T cell subsets and antibody production during the course of infection.

The C57Bl/6 susceptible (Bcgs) and its resistant (Bcgr) congenic mouse, previously developed by retrogressive backcrossing, were infected with 1 x 10(6) colony-forming units (CFU) of Mycobacterium avium and bacterial growth and their immune responses during the early and prolonged periods of infection were examined. There was a high proliferation in the liver and spleen of Bcgs mice, whereas no proliferation was observed in the Bcgr mice. Similarly, the sizes and weights of these organs were much higher than those of their Bcgr counterparts. The size and number of granulomas in Bcgs were also found to be higher than those of Bcgr. The CD3+ and CD4+ subsets increased dramatically in both mice during the early stage of infection. However, in the later phase of the infection, these populations decreased dramatically in Bcgs mice, but not in Bcgr mice, resulting in a depression in cell-mediated immune responses. No significant decrease in cell-mediated immune responses was observed in Bcgr mice even after prolonged infection. ELISA was performed to determine the antibody levels in both mice, and it was found that serum IgG and IgM levels in Bcgs were comparatively higher than those in Bcgr mice throughout the period of infection. The Bcg gene therefore may have an important role in the maintenance of resistance not only in the early phase but also in the later phase of Myco. avium infection.

Granulomatous epididymitis related to Rhodotorula glutinis infection in a dog.

A 4-year-old, male Great Dane dog developed severe swelling of the scrotum on 9 December 1991, and the testes and epididymides were removed surgically on 12 December 1992. The cut surface of the epididymides consisted of hard connective tissue and several small abcesses with slight hemorrhage. Histopathologically, the seminiferous tubules in the testes had only a few spermatogenic cells, but Sertoli cells were well preserved. Both epididymides consisted entirely of a proliferation of fibrous connective tissue, and only a few ducts deferens containing cell debris, neutrophils, and macrophages in the lumina were present. In all lesions of the epididymides, the macrophages contained periodic acid-Schiff- and Grocott's silver-positive round granules, 5-8 microns in diameter. Microbiologically, smooth salmon-pink colonies consisting of ovoidal yeast, about 10 microns in diameter, were isolated from the samples of epididymides but not from those of the testes. The isolated yeast had microbiological characteristics of Rhodotorula glutinis. From these observations, we diagnosed the present case as granulomatous epididymitis due to Rhodotorula infection.

Cardiovascular system related peptides and hypothalamic neurons.

The hypothalamus is known to be an integrative site of cardiovascular, endocrine and autonomic functions. Our previous studies, using extracellular, intracellular and/or whole cell patch-clamp recordings in rat hypothalamic slice preparations, revealed that cardiovascular related peptides such as atrial natriuretic polypeptides (ANP), B-type polypeptides (BNP), endothelin (ET), angiotensin II (AII) and interleukin-1 beta (IL-1 beta) influence the hypothalamic neurons. ANP modulated the firing rates in the supraoptic nucleus (SON). BNP inhibited the SON neurons and these effects were mediated through cGMP and cGMP-dependent protein kinase. ET also inhibited approximately 60% of SON neurons. By using slice patch-clamp techniques, AII inhibited the transient outward potassium current in the SON neurons. IL-1 beta increased the firing rate and depolarized the membrane of the most SON neurons. A new type of transmitter, nitric oxide (NO), identified as an endothelial-derived relaxing factor (EDRF), modulated the glutaminergic inputs of the SON neurons. The results suggest that cardiovascular related peptides and NO modulate the neuronal activity of neurosecretory cells in the SON.

The sequence and correlation of the changes in several parameters of energy metabolism induced by ischemic insult in heart muscle.

Samples of myocardium with varying degrees of ischemia were obtained from subendo- and subepicardium of nonischemic, ischemic border and center zones of canine left ventricles to assess the sequence of rapid alterations of several parameters of energy metabolism induced by myocardial ischemia. Ischemia was induced by occluding the left anterior descending coronary artery. The contents of creatine phosphate (CP) and ATP decreased significantly. There was a significant accumulation of lactate. The redox potential (Eh) and phosphorylation potential (l') also decreased sharply, but only a slight decrease in energy charge (E.C.) was noted. A close correlation was found between ATP and total adenine nucleotide (r = 0.99). There were various degrees of linear correlation between other parameters, except for ATP (and total adenine nucleotide) and Pi, and CP and l', which were uncorrelated. The sequence of rapid alterations in the parameters of energy metabolism induced by ischemia was: (1) l' and CP, (2) Pi, ATP and Eh, and finally (3) lactate and E.C. A significant increase of the heart rate and a decline of the mean blood pressure and left ventricular pressure (LVP) were also observed in association with a slight change in the maximum rate of rise of the left ventricular pressure (dP/dtmax).