Voltage-dependent Ca2+ channels are involved in regulation of steroid synthesis by bovine but not rat fasciculata cells.

Bovine but not rat fasciculata cells show a concentration-dependent stimulation of the production of corticosteroids by addition of external Ca2+ to the incubation medium. Both cell types respond to ACTH in a concentration-dependent manner. Increasing concentrations of K+ (0-20 mM) cause increased production of corticosteroids and accelerated influx of Ca2+ in bovine fasciculata cells but no change in either of these two parameters in rat fasciculata cells. Two inhibitors of Ca2+ channels (nifedipine and PY108-068) inhibit both the production of steroids by unstimulated bovine cells and the stimulation produced by three agents (ACTH, (Bu)2 cAMP, and K+). Half-maximal inhibition of these responses was produced in each case by submicromolar or low micromolar concentrations of the inhibitors. These inhibitors are without effect in rat fasciculata cells. One Ca2(+)-channel agonist (BAY K8644) stimulated synthesis of steroids by bovine cells and potentiated the response to ACTH. The agonist acts in the low micromolar range but was without effect on rat cells or on the responses of steroid synthesis by these cells to either ACTH or dibutyryl cAMP. Moreover, bovine fasciculata cells show specific binding sites for [+]PN 200-110, a specific ligand for 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels [dissociation constant (Kd), 14.3 nM; maximum number of binding sites (Bmax), 0.52 pmol/10(6) cells]. Rat cells show no specific binding of PN 200-110. We conclude that bovine fasciculata cells possess voltage-dependent Ca2+ channels which are involved in the regulation of steroid synthesis in these cells and that rat fasciculata cells are without such channels.

The regulation of intracellular transport of cholesterol in bovine adrenal cells: purification of a novel protein.

We have purified to homogeneity a protein from bovine adrenal (fasciculata) cells that is capable of: 1) stimulating synthesis of pregnenolone (in a concentration-dependent fashion) by mitochondria when added with exogenous cholesterol; 2) increasing the concentration of cholesterol in outer and inner membrane when added to mitochondria with exogenous cholesterol; 3) increasing the rate of transport of cholesterol from outer membrane to inner membrane when the two membranes are incubated together in an aqueous buffer and subsequently separated by centrifugation; and 4) increasing the proportion of molecules of C27 side-chain cleavage P-450 that are bound to the substrate cholesterol. The protein is homogeneous according to two-dimensional polyacrylamide gels, shows a molecular weight of 8200 and is therefore referred to as 8.2 K. It is proposed that 8.2 K may be involved in the regulation of steroid synthesis in bovine fasciculata cells, possibly by stimulating entry of cholesterol into the outer membrane and by altering the intramitochondrial distribution of this substrate.

Forskolin activates voltage-dependent Ca2+ channels in bovine but not in rat fasciculata cells.

The action of forskolin on bovine and rat fasciculata cells was examined in freshly prepared cells. Bovine cells show a close parallelism between production of steroids and production of cAMP as a function of the concentration of ACTH up to 10(-8) M. By contrast, forskolin (10(-7)-10(-5) M) causes a similar increase in steroid synthesis but relatively little effect on the production of cAMP. cAMP-dependent protein kinase shows a similar response to ACTH but no response to forskolin in the same range of concentrations. ACTH and forskolin, at submaximal concentrations, cause greater steroid production when added together than when added separately, but the two agents at high concentrations produce the same response whether added together or separately. The inhibitors of voltage-dependent Ca2+ channels inhibit the steroidogenic response to forskolin (IC50 for nifedipine is 0.1 microM and for Py108-068 is 0.4 microM). A Ca2+ channel agonist (BAY K8644) increases the steroidogenic response of bovine adrenal cells to forskolin, but not that of ACTH. Finally, forskolin causes a concentration-dependent uptake of Ca2+ by these cells; in the concentration range of 0.1-10 microM, forskolin caused an increase in [Ca2+] from 185 nM to 345 nM. By contrast, forskolin caused some stimulation of the production of cAMP, but not that of steroids in rat fasciculata cells. It is concluded that in bovine fasciculata cells forskolin activates voltage-dependent Ca2+ channels with a consequent increase in steroid synthesis. This effect is independent of the well known action of forskolin on adenylate cyclase. Rat fasciculata cells, on the other hand, do not possess such Ca2+ channels and do not show a steroidogenic response to forskolin.

Preparation and characterization of submitochondrial fractions from adrenal cells.

A method for preparing submitochondrial fractions from adrenocortical cells was developed by adapting a procedure that has been successful with yeast mitochondria. The method is based upon osmotic swelling, sonication and centrifugation in sucrose. The preparation yields highly purified fractions of outer membrane, intermembrane space, inner membrane and a less purified fraction of matrix. Recoveries are good so that 10(7) cells yield approximately 170 micrograms of inner membrane protein and 12 micrograms of outer membrane protein. Electron microscopy shows that the outer membrane fraction consists of vesicles (0.2-0.6 micron diameter) while inner membrane appears as densely packed sheets of membranous material. Two-dimensional polyacrylamide gels (isoelectric focusing followed by electrophoresis) of all the fractions give highly reproducible patterns of protein spots with Coomassie staining. Steroidogenic proteins were found only in inner membrane fractions which were shown to contain cytochrome P-450 C27 side-chain cleavage and P-450 11 beta-hydroxylase together with adrenodoxin and adrenodoxin reductase. Inner membrane catalyzes side-chain cleavage of cholesterol (conversions to pregnenolone) and 11 beta-hydroxylation (DOC----corticosterone) when substrate and NADPH are added. The preparation yields highly purified submitochondrial fractions from Y-1 mouse adrenal tumor cells and from porcine and bovine adrenocortical mitochondria. The method has the virtue of yielding highly purified intermembrane fluid which is not true of other methods for fractionation of adrenal mitochondria. The procedure also yields cleaner preparations of the two membranes than two other published methods currently used to prepare submitochondrial fractions from adrenocortical cells.

Protein rotation study of cytochrome P-450 in submitochondrial particles: effect of KCl and intermolecular interactions with redox partners.

The rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate the effect of KCl on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KCl concentration. When the KCl concentration was increased from 0 to 1,000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCl, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KCl, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cytochrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-450SCC forms a transient ternary association with adrenodoxin and adrenodoxin reductase.

Conversion of cholesterol to pregnenolone mobilizes cytochrome P-450 in the inner membrane of adrenocortical mitochondria: protein rotation study.

Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria.

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.

Postoperative radiation therapy for esophageal cancer.

The value of postoperative radiation therapy (RT) was investigated in 77 patients with esophageal cancer resected between 1977 and 1986. Surgical resection was palliative in 13 of these patients. Although seven of them underwent postoperative irradiation to the residual tumor, all of the patients died within one year. Following potentially curative resection performed in 64 patients, 31 patients received 50 Gy of postoperative RT to the lower neck and the mediastinum (group Ia), seven were unable to receive full-dose postoperative RT (group Ib), and 26 were not treated with postoperative RT (group II). The 5-year survival rate estimated by the Kaplan-Meier method was 54% for group Ia, 29% for group Ib, and 33% for group II, with the difference between groups Ia and II being significant (p less than 0.025). The local recurrence rate in the mediastinum was lower in group Ia than in group II. Prophylactic postoperative RT for esophageal cancer is a safe and effective regimen for patients with resected disease.

[Surgical treatment of hemifacial spasm associated with tortuous vertebrobasilar system (author's transl)].

Surgical treatment of hemifacial spasm associated with tortuous vertebrobasilar system was reported. A patient was 63-year-old female, who first experienced mild and intermittent muscle twitching around her left eye twenty years prior to admission. Five years later, the twitching extended to all the facial muscles on the left side. She was treated with facial nerve block, which resulted in facial palsy for about one year. Because of recurrence of the hemifacial spasm, she was admitted to the Neurosurgical Department of Bokuto Municipal Hospital on October 12, 1977. Neurological examination revealed no abnormalities except for left hemifacial spasm with slight muscular weakness. Electromyogram showed severe twitching and synkinesis of all the muscles of facial expression. Vertebral angiogram on the left side disclosed pronounced elongation of the vertebral and basilar arteries, which extended into the left cerebellopontine angle. Compression of the facial nerve root exit zone at the brainstem by the vertebral artery was considered to be the cause of the hemifacial spasm. Suboccipital craniectomy was carried out on November 29, 1977. The vertebral artery extended into the cerebellopontine angle, and adhered to the facial nerve. After mobilization of the vertebral artery from the facial nerve, a small prosthesis of non-absorbable spongy material (Teflon felt) was interposed between the vertebral artery and brainstem. Postoperatively, the hemifacial spasm disappeared, but the facial palsy, which had been observed preoperatively probably due to previous facial nerve block and long-standing hemifacial spasm, remained. The function of the acoustic nerve was preserved. Recently vascular compression of the facial nerve root exit zone has been reported as a major cause of hemifacial spasm, but such abnormal vessels are rarely demonstrated angiographically.

[Hemangioblastoma of the cauda equina--a case report (author's transl)].

We reported the case of a 40-year-old man who was hospitalized to our department on November 16, 1975 with a year history of neuralgia in the saddle region and vesicorectal dysfunction. Examination of CSF on lumbar puncture at L4-5 revealed xanthochromia with a protein content of 2,560 mg/dl. Myelography revealed a tortuous filling defect at the level of L2 coupled with complete blockade at L3 level. Selective spinal angiography of the left 12th intercostal artery demonstrated the enlarged Adamkiewicz's artery. This blood vessel was found to enter a diffuse uniform density, where no distinct blood vessels were visible. Within one second after the injection of contrast agent an efferent vein appeared from the right side of the density, ascended to the level of Th12 and then, upon turning, descended to the pelvic canal. These findings suggested a hemangioblastoma of the spinal cord. Laminectomy was performed at levels of L2 through L4 and a hemangioblastoma of the cauda equina was totally excised at L3 level. Although the postoperative course was complicated by a transient dysuria and hypesthesia at S1-S5 levels, complete cure was achieved in a month. In the present paper clinical features and neuroradiological examination of a hemangioblastoma of the spinal cord were described.

[Spontaneous regression of sinus pericranii--report of a case (author's transl)].

Spontaneous regression of sinus pericranii has not been reported in the literatures. The aurthors presented a very rare case of sinus pericranii, which was diagnosed as early as nine days after birth and completely disappeared in seven years. The patient was a nine-day-old boy from normal delivery in full term, who had a soft tumor simulating cephalhematoma in the right parietal region. The overlying scalp looked like a hemangioma bitemporally connecting with strikingly dilated scalp veins. Soft X-ray examination of the skull revealed a homogenous mass and a small bone defect beneath the mass. Venous blood was punctured from the tumor. By means of direct injection of contrast media into the tumor, the superior sagittal sinus as well as many extracranial varicositous veins were shown. No intracranial vascular anomaly. In a year the tumor spontaneously declined and in the following seven years it disappeared completely. Pathogenesis of this lesion was discussed with special reference to cephalhematoma and a possibility of spontaneous regression of sinus pericranii was stressed.