C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro

Abstract

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.

Fig. 1

Sections of pons of the rat labeled with C-INSL7 antiserum or C-INSL7 antiserum pre-absorbed with the C-INSL7 peptide (1μg/kg) overnight. A, irC-INSL7 neurons are distributed to the nucleus incertus pars compacta (NIc) and nucleus incertus pars dissipata (NId); they appear multipolar with extensive branching. B, a pontine section, at a level similar to that shown in panel A, labeled with C-INSL7 antiserum pre-absorbed with the peptide (1 μg/ml) overnight; irC-INSL7 neurons are absent in the nucleus incertus. C and D, sections of pons where few irC-INSL7 neurons are noted in the lateral periaqueductal gray (LPAG) and ventrolateral periaqueductal gray (VLPAG); they appear bipolar, with few branching. Abbreviations: 4V, 4th ventricle; Aq, aqueduct; DTg, dorsal tegmental nucleus. Scale bar: A–D, 100 μm.

Fig. 2

Fluorescence images of rat forebrain sections labeled with C-INSL7 antiserum A–C, in the septum, a dense network of irC-INSL7 fibers is seen in the lateral septum nucleus, intermediate (LSI) and medial septum (MS); a moderate density of irC-INSL7 fibers is present in lateral septum nucleus, ventral (LSV), and nucleus of vertical limb of the diagonal band (VDB). D, irC-INSL7 fibers are present in the bed nucleus of stria terminalis, medial division, anterior (BSTMA) and ventral division (BSTV). E–L, varying intensities of irC-INSL7 fibers are present in several hypothalamic nuclei such as: medial preoptic area (MPA), lateral preoptic area (LPO), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LH), ventromedial hypothalamic nucleus (VMH), periventricular hypothalamic nucleus (Pe), dorsal hypothalamic area (DA) and dorsomedial hypothalamic nucleus (DMD), and tuber cinereum (TC). M–N, irC-INSL7 fibers were present in lateral habenular nucleus (LHb), paraventricular thalamic nucleus, anterior (PVA) and scarce in the medial habenular nucleus (MHb). O–P, irC-INSL7 fibers in endopeduncular nucleus (EP) and basolateral amygdaloid nucleus, anterior (BLA). Abbreviations: aca, anterior commissure, anterior; 3V, 3^rd ventricle; D3V, dorsal 3^rd ventricle; opt, optic tract; sox; supraoptic decussation. Scale bar, 50 μm.

Fig. 3

Elevation of cytosolic Ca²⁺ concentrations in cultured hypothalamic neurons by INSL7 and C-INSL7. A, INSL7 (100 nM) produced a fast and transitory increase in [Ca²⁺]_i in this neuron; the mean [Ca²⁺]_i increase was 817 ± 6.3 nM (n=27). B, A fast response followed by calcium oscillations was evoked by INSL7 (100 nM) in 8 neurons; the mean amplitude of the first spike was 1268 ± 39 nM. C, C-INSL7 (10, 100 and 1000 nM) produced a fast and sustained increase in [Ca²⁺]_i in a concentration-dependent manner: the mean [Ca²⁺]_i increase was 118 + 2.4 nM (n = 6) for 10 nM C-INSL7 (green trace), 405 + 3.1nM (n=36) for 100 nM (black trace) and 731 + 4.7 nM (n = 8) for 1000 nM C-INSL7 (red trace) respectively. D, C-INSL7 induced calcium oscillations also in a concentration-dependent manner: the mean amplitude of the first spike was 137 + 2.6 nM (n = 6) for 10 nM (green), 763 + 4.2 nM (n = 11) for 100 nM (black) and 1051 + 9.8 nM (n = 7) for 1000 nM, respectively (red). E, pretreatment with ryanodine (5 μM; 15 min) had no significant effect on C-INSL7 (100 nM)-induced fast and sustained increase in [Ca²⁺]_i (Δ [Ca²⁺]_i = 391 + 3.6 nM; n = 7). F, pretreatment with ryanodine abolished [Ca²⁺]_ioscillation, C-INSL7 (100nM) caused a fast, transitory increase of [Ca2+]i by 427 + 3.2 nM (n = 9).

Fig. 4

Calcium responses produced by INSL7 and C-INSL7 in Ca²⁺-free saline. A, INSL7 (100 nM) produced a rapid and transitory increase in [Ca²⁺]_i by 307 ± 4.2 nM (n=11). B, INSL7 induced [Ca²⁺]_i oscillations with a mean amplitude of the first spike of 583 ± 7.4 nM (n=5). C, In this neuron, C-INSL7 (100 nM) produced a fast and transitory increase in [Ca²⁺]_i; the mean amplitude of [Ca²⁺]_i spike was 231 ± 2.6 nM (n=15). D, C-INSL7 induced [Ca²⁺]_i oscillations with a mean amplitude of the first spike of 497 ± 3.6 nM (n=7).

Fig. 5

Optical imaging of membrane potential changes by C-INSL7 in dissociated rat hypothalamic neurons. A, C-INSL7 depolarized (top trace) or hyperpolarized (bottom trace) hypothalamic neurons. B, the mean amplitude of depolarizations was 11 ± 1.6mV (n=7) and the mean amplitude of hyperpolarizations was 7 ± 1.3mV (n=15).

Figure 6

C-INSL7 binding in rat brain membranes: [¹²⁵I]C-INSL binding was performed in the presence or absence of various concentrations of unlabeled C-INSL7 and INSL7 as described in Experimental procedures. Data are expressed as mean ± s.e.m. of three independent experiments.