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Biosynthesis and release of VIP-related peptides in a human neuroblastoma cell line
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Biosynthesis and Release of VIP-Related Peptides in a Human Neuroblastoma Cell Line M. HOSHINO*, Y. HIOKI*#, C. YANAIHARA#, K. OGINOt, T. SUZUKI* and N. YANAIHARA*t (*National Institute for Physiological Sciences, Okazaki, and tShizuoka College of Pharmacy, Shizuoka, Japan) The production and release of VIP-like immunoreactivity in human neuroblastoma NB-OK-I cells were stimulated by the addition of (But)2cAMP. In the stimulated condition, [14-C]Leu was incorporated during 4 - 15 hr incubation into VIP-like immunoreactive components in the cells. The radiolabeled components were shown, by SDSpolyacrylamide gel electrophoresis after immunoaffinity chromatography, to comprise four major molecular forms of 3K, 8K, IIK and 18K daltons (d). On prolonging the labeling period, the 18Kd-component increased markedly, while the increase of the 3Kd-component was relatively small. The three larger forms, 8Kd-, llKd- and 18Kdcomponents in the cells almost disappeared after 4 hr chase-labeling. During the incubation with [14-C]Leu, radiolabeled VIP-like immunoreactive materials were also found in the culture medium, which contained three major components of 3Kd, 8Kd and 18Kd. The 3Kd-component in the medium increased markedly on prolongation of labeling period and the 8Kd- and 18Kd-components also increased to a lesser extent. The immunoreactive 3Kd-component was isolated from the ~e]1 extract by a combination of gel filtration, immunoaffinity chromatography and ~PLr, and it was found to contain four molecular species including the one behaving like porcine VIP on HPLC. On the other hand, the extract of the tumour, which was ~or~ed by implanting the NB-OK-I cells intraperitoneally or subcutaneously in nude mice, was shown by gel filtration and HPLC to contain porcine VIP-like component as a major constituent of VIP-llke irm~unoreactivity with very minor larger molecular-size components. These results indicate that the immunoreactlve 3Kd-component is formed in the NB-OK-I cells from a larger molecular form and that not only the 3Kd-component but also the VIP-like immunoreactive components of larger molecular forms are apparently released from the cultured cells. The present study also suggests possible difference in processing of the VIP precursor in the NB-OK-I cells and in the tumour produced by implantation of the cells in nude mice.
Acute Stimulation of Ganglionic Tyrosine Hydroxylase Activity by Secretin~ Vasoactive Intestinal Peptide and PHI. N.Y. IP and R.E. ZIGMOND, (Department of Pharmacology, Harvard Medical School, Boston, MA 02115 USA) We previously reported that stimulation of the preganglionic cervical sympathetic trunk leads to an acute increase in tyroslne hydroxylase (TH) activity in the rat superior cervical ganglion (SCG). This increase appears to be mediated in part by acetylcholine and in part by a non-cholinergic transmitter. In an attempt to identify this transmitter, we screened a number of neuropeptides at a concentration of i0 ~M for their ability to increase TH activity. Angiotensin II, bombesin, bradyklnin, cholecystokinin octapeptlde, insulin, luteinizing hormone-releasing hormone, [D-Ala2,Met 5] enkephallnamide, motilin, neurotensin, somatostatin and substance P produced no effects. Secretin and vasoactive intestinal peptide (VIP) were found to increase TH activity with an EC50 of 5 nM and 0.5 ~M, respectively. The effects of these peptides were not altered by prior decentralization of the ganglia, by addition of hexamethonium (3 mM) and atropine (6 ~M), or by lowering the concentration of calcium in the medium to 0.i mM. Addition of carbachol (3 ~M) potentiated the effects of both secretin and VIP on TH activity. Several gastrointestinal peptides with structural similarities to secretin and VlP were examined for their ability to increase TH activity. Glucagon and gastric inhibitory peptide produced no effect at a concentration of i0 ~M, while PHI increased enzyme activity. The EC50 of this effect of PHI was about 3 ~M. Whether secretin, VlP or PHI mediates the non-cholinerglc increase in TH activity following preganglionic nerve stimulation in the SCG remains to be determined.
Biosynthesis and Release of VIP-Related Peptides in a Human Neuroblastoma Cell Line M. HOSHINO*, Y. HIOKI*#, C. YANAIHARA#, K. OGINOt, T. SUZUKI* and N. YANAIHARA*t (*National Institute for Physiological Sciences, Okazaki, and tShizuoka College of Pharmacy, Shizuoka, Japan) The production and release of VIP-like immunoreactivity in human neuroblastoma NB-OK-I cells were stimulated by the addition of (But)2cAMP. In the stimulated condition, [14-C]Leu was incorporated during 4 - 15 hr incubation into VIP-like immunoreactive components in the cells. The radiolabeled components were shown, by SDSpolyacrylamide gel electrophoresis after immunoaffinity chromatography, to comprise four major molecular forms of 3K, 8K, IIK and 18K daltons (d). On prolonging the labeling period, the 18Kd-component increased markedly, while the increase of the 3Kd-component was relatively small. The three larger forms, 8Kd-, llKd- and 18Kdcomponents in the cells almost disappeared after 4 hr chase-labeling. During the incubation with [14-C]Leu, radiolabeled VIP-like immunoreactive materials were also found in the culture medium, which contained three major components of 3Kd, 8Kd and 18Kd. The 3Kd-component in the medium increased markedly on prolongation of labeling period and the 8Kd- and 18Kd-components also increased to a lesser extent. The immunoreactive 3Kd-component was isolated from the ~e]1 extract by a combination of gel filtration, immunoaffinity chromatography and ~PLr, and it was found to contain four molecular species including the one behaving like porcine VIP on HPLC. On the other hand, the extract of the tumour, which was ~or~ed by implanting the NB-OK-I cells intraperitoneally or subcutaneously in nude mice, was shown by gel filtration and HPLC to contain porcine VIP-like component as a major constituent of VIP-llke irm~unoreactivity with very minor larger molecular-size components. These results indicate that the immunoreactlve 3Kd-component is formed in the NB-OK-I cells from a larger molecular form and that not only the 3Kd-component but also the VIP-like immunoreactive components of larger molecular forms are apparently released from the cultured cells. The present study also suggests possible difference in processing of the VIP precursor in the NB-OK-I cells and in the tumour produced by implantation of the cells in nude mice.
Acute Stimulation of Ganglionic Tyrosine Hydroxylase Activity by Secretin~ Vasoactive Intestinal Peptide and PHI. N.Y. IP and R.E. ZIGMOND, (Department of Pharmacology, Harvard Medical School, Boston, MA 02115 USA) We previously reported that stimulation of the preganglionic cervical sympathetic trunk leads to an acute increase in tyroslne hydroxylase (TH) activity in the rat superior cervical ganglion (SCG). This increase appears to be mediated in part by acetylcholine and in part by a non-cholinergic transmitter. In an attempt to identify this transmitter, we screened a number of neuropeptides at a concentration of i0 ~M for their ability to increase TH activity. Angiotensin II, bombesin, bradyklnin, cholecystokinin octapeptlde, insulin, luteinizing hormone-releasing hormone, [D-Ala2,Met 5] enkephallnamide, motilin, neurotensin, somatostatin and substance P produced no effects. Secretin and vasoactive intestinal peptide (VIP) were found to increase TH activity with an EC50 of 5 nM and 0.5 ~M, respectively. The effects of these peptides were not altered by prior decentralization of the ganglia, by addition of hexamethonium (3 mM) and atropine (6 ~M), or by lowering the concentration of calcium in the medium to 0.i mM. Addition of carbachol (3 ~M) potentiated the effects of both secretin and VIP on TH activity. Several gastrointestinal peptides with structural similarities to secretin and VlP were examined for their ability to increase TH activity. Glucagon and gastric inhibitory peptide produced no effect at a concentration of i0 ~M, while PHI increased enzyme activity. The EC50 of this effect of PHI was about 3 ~M. Whether secretin, VlP or PHI mediates the non-cholinerglc increase in TH activity following preganglionic nerve stimulation in the SCG remains to be determined.