C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.
With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.
Brailoiu
E
E
Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Dun
S L
SL
Gao
X
X
Brailoiu
G C
GC
Li
J-G
JG
Luo
J J
JJ
Yang
J
J
Chang
J K
JK
Liu-Chen
L-Y
LY
Dun
N J
NJ
D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D002096:C-Peptide / Q000378:metabolism* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology / Q000379:methods; D016257:Fura-2 / Q000378:metabolism; D007031:Hypothalamus / Q000166:cytology; D007456:Iodine Isotopes / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013858:Thiobarbiturates / Q000378:metabolism
D002096:C-Peptide; D007456:Iodine Isotopes; D013858:Thiobarbiturates; C106875:bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol; D002118:Calcium; D016257:Fura-2