Atrial natriuretic peptide-like immunoreactive cells in the guinea pig inner ear

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Atria1 natriuretic peptide-like immunoreactive inner ear A.M. Meyer zum Gottesberge, M. Gagelmann

’

cells in the guinea pig

and W.G. Forssmann ’

Forschungslabor der HNO Klinik der lJniL:ersitiitDiisseldorf, ’ Institut fiir Anatomie und Zellbiologie, UniL:ersitiitHeidelberg, 2 Niedersiichsisches Institut fiir Peptid-Forschung GmbH (IPF), Hannwer, F.R.G. (Received

I2 December

1990; accepted

2X April

1991)

Using specific antibodies against cardiodilatin/atrial natriuretic peptide (CDD/ANP) in a conventional immuno-histochemical method (PAP) we located ANP/CDD-like immuno-reactive cells related to the secretory area, to the sensory and to the neuronal area in the compartments of the inner ear (cochlea, utricle/ampulla, and endolymphatic sac). Immunoreactive cells were unevenly distributed in the different compartments as well as within the cochlear space. Our findings suggest that ANP/CDD may play a role in the local control of fluid and electrolyte homeostasis of the inner ear. ANP/CDD-binding sites and ANP/CDD-like immunoreactivity in the inner ear may also indicate that the peptide has an additional paracrine and/or autocrine function in the organ. Atrial

natriuretic

peptides;

Cardiodilatin:

Hormonal

regulation;

Cochlear

Introduction

Atria1 natriuretic peptides (ANP) are a group of bioactive peptides with potent natriuretic, diuretic and vasodilating effects which were first discovered and isolated from mammalian atria in the early 1980s (Brenner et al., 1990; Forssmann, 1986; Genest and Cantin, 1988). Myocardiocytes appear to be the major site of synthesis and secretion. Immunohistochemical activity of ANP however, was observed in other tissues such as brain, peripheral nervous system, salivary glands, lacrimal glands as well as in the anterior pituitary gland and the adrenal medulla (Genest and Cantin, 1988). These extracardial sites of ANP-like synthesis suggest that the peptide has additional peripheral paracrine or autocrine functions (Forssmann et al., 1989). ANP acts on specific receptors activating particulate guanylate cyclase and hence increases intracellular messenger cyclic guanosine 3’, 5’-monophosphate (cGMP) levels in the target tissue (Brenner et al., 1990; Genest and Cantin, 1988). ANP receptors have been demonstrated in various tissues mainly responsible for maintaining fluid and electrolyte homeostasis such as kidney, adrenal gland, ciliary body, blood vessels, the chorioid plexus of the cerebral ventricles (Genest and Cantin, 1988) and recently also in the inner ear Correspondence to: A.M. Meyer zum Gottesberge, Forschungslabor der HNO Klinik, Heindrich-Heine-Universitlt Dusseldorf, Moorenstrasse 5, 4000 Dusseldorf, F.R.G.

duct; Vestibular

organ;

Outer

hair cells

(Lamprecht and Meyer zum Gottesberge 1988). Using labelled ANP and autoradiographic methods we were able to demonstrate diffuse distribution of specific binding sites in the cochlea with intense labelling in the sublateral region of the cochlear lateral wall and the vessels of the modiolus. The aim of our present investigation, extending preliminary observations (Meyer zum Gottesberge et al., 1989), was to investigate the presence of ANP/CDDreactive cells in the inner ear of normal guinea pigs. Using immunohistochemical techniques and antibodies against atria1 natriuretic peptides/cardiodilatin (ANP/ CDD> (Forssman et al., 1983) we report here on the distribution of this peptide in the cochlea, the utricle/ ampulla compartment and in the rugosal and distal part of the endolymphatic sac. Material and Methods

10 pigmented guinea pigs (males), weighing about 300 g and showing positive Preyer’s reflex were used. The intracardial perfusion with saline and Bouin’s fixative was performed in sodium pentobarbital anesthesia (Nembutal 30 mg/kg, b.w., i.p.>. Temporal bone and adjacent lining of the posterior cranial fossa were excised en bloc, immersed in fixative over night at 4°C dissected and embedded in paraffin. Immunohistochemical localization of the peptide was carried out on 3 Frn paraffin sections using the peroxidase-antiperoxidase method (PAP), using 3,3_diaminobenzidine as substrate. Several polyclonal antisera raised against

87

ANP/CDD-99-126 (cu-hANP) in rabbit, were diluted 1: 1000 or 1: 2000 with PBS with 1% swine serum and incubated with the sections for 56-72 h at 4°C. Secondary antibody against rabbit IgG (Dakopatts) were diluted 1: 50. The immunoreaction was visualized using peroxidase and rabbit antiperoxidase (Dakopatts). Omission of primary antibody completely suppressed the staining of the tested section (negative control); sections of swine atria were used as a positive control.

For proof of the specificity the antiserum was absorbed with lop6 M ANP/CDD-99-126 for 12 h.

Results Several cell populations of the various compartments of the inner ear show positive immunoreaction for ANP/CDD-like peptides. The animals exhibited

3-turn

a

a

Fig. 1 (a) Cochlear lateral wall of the different turns stained for ANP/CDD and (b) after preabsorption with lo-6M ah-ANP/CDD-99-126 for 12 h, 50X (detail in higher magnification as Fig. 8). SC= stroma cells of the spiral ligament, sv = stria vascularis, rcb = root cell bundle, cc = Claudius cells, m = melanin

xx

individual variability in the intensity of the immunolabelling within identical cells. In the cochlear lateral wall the stroma cells of the spiral ligament, root cells, the cells of the external sulcus and Claudius cells were stained. Differences were found in the number of immunopositive cells in the individual turns. Most immunoreactive cells were found in the basal turn and the number of positive cells decreased toward the apical turn (Fig. la). In contradistinction, the cells of the stria vascularis of lower turns were not stained with the exception of several individual granules near the capillaries (Fig. 8). The presence of granules increased in the apical turn. The

Fig. 2. (a) lmmunohistochemical staining absence of ANP/CDD-immunnoreactivity

Fig. 3 (a) lmmunohistochemical

immunoreaction of the lateral wall was almost completely reduced after preabsorption of the antibodies with 1Op6M ANP/CDD-99-126 (Fig. lb). In the organ of Corti the outer hair cells, the supporting cells-, Deiter’s, Hensen, and pillar cells - and the cells of the internal sulcus were stained with the exception of the inner hair cells (Fig. 2a). Several spiral ganglion cells (Fig. 3a), located mostly laterally, showed stronger labelling. This reaction was absent after absorption with the specific antigen. The same absorption however, produced only suppression of the immunohistochemical reaction in other spiral ganglion cells and in the organ of Corti (Fig. 2b and 3b)

of ANP/CDD of the organ of Corti (apical turn) and (b) after preabsorption with antigene; note the in the inner hair cell, 170x., inset in (a): inner hair cell region, 255X ohc = outer hair cells, ihc = inner hair cell

staining with ANP/CDD of laterally located cells of the spiral ganglion antigene, 325 X: arrows indicate granular staining of the cytoplasm

and (b) after

preabsorption

with

89

No positive cells were identified in the secretory area of the vestibular organ (Fig. 4). In the sensory areas the reaction was positive in hair cell calyces and was partly suppressed by absorption. In the endolymphatic sac (Fig. 5) most labelled cells were found in the epithelial layer of the pars rugosa, whereas in the distal part only single cells were stained. Absorption with ANP/CDD-99-126 produced suppression of the reaction only in some cells. Strong unspecific labelling was observed in some individual cells (probably plasma cells) and free floating cells in the lumen of the endolymphatic sac.

Discussion Using specific antibodies against cardiac peptides (ANP/CDD-99-126 = aANP) we were able to identify several cell populations of the inner ear which were related to i) the secretory area, ii> the sensory area, and iii) the neuronal area. In contrast to the secretory area (cochlear lateral wall) the immunopositive cell population of the sensory and neuronal areas of the cochlea showed identical reaction in all cochlear turns. In the cochlear lateral wall a decrease in the number of positive cells was

. \

,._.’ .

‘.

. 8

b

Figs. 4-6. Consecutive sections of the inner ear showing different intensity of immunostaining against ANP/CDD (a) and against antibody absorbed previously with ah-ANP/CDD-99-126 for 12 h (b); arrows indicate cells with positive reaction. Fig. 4. crista ampullaris, 50x, inset = receptor area of high magnification. Fig. 5. rugosal part of the endolymphatic sac, 200X. Fig. 6. atria1 tissue (transversly sectioned myocardic cells) serving as positive control, 200 x ; ca = crista ampullaris, m = melanin.

00

noticed towards the apical turn. Furthermore, an uneven distribution of ANP/CDD-like immunoreactivity was observed between the cochlear and vestibular (utricle/ampulla) secretory compartments. The localization of the ANP/CDD binding sites in the inner ear corresponds to the areas known to play a major role in the maintenance of fluid/electrolyte homeostasis in the inner ear (Meyer zum Gottesberge and Lamprecht, 1989). Therefore an autocrine and paracrine function of ANP/CDD in the inner ear may be postulated. In 1965 Wilding described the presence of chromafin cells in the lateral wall of the cochlea which mostly correspond to locations of ANP/~DD-Iike immunoreactive cells. These cells are located in the connective tissue beneath the epithelium of the stria vascularis and in the spirai prominentia in close proximity to the blood vessels in a so-called ‘strategic anatomical area’ (Hilding, 196.5). This author speculated that these cells may release their products into the surrounding area and may affect local blood flow, hence they would influence the production of the endolymph via stria celIs. Furthermore, they would be advantageously placed to monitor both, endolymph and perilymph. Since the role of CAMP in electrolyte transport in the tubular membranes of cyclase activity in the kidney was established, the demonstration of adenylate cyclase activity in inner ear tissue (in vitro) has been of great interest in studying cochlear physiolo~ (Koch

Fig. 7. ANP/CDD-like

immunoreactivity

frcb). sp = spiral prominentia,

of the root cell bundle 180X.

Fig. 8. Detail of cochlear lateral wall of the basal turn of Fig. la. 260x. sv = stria vascularis, SC= stroma cells of the spiral ligament, c = capillary

and Zenner 1988; Schacht, 1985; ~almann et al.,). Physiological response of the inner ear, confirmed by electrophysiological studies (Mori et al., 19891, indicates that hormones involved in the regulation of electrolyte and fluid homeostasis in the body as a whole also affect inner ear function. There is now conclusive evidence that the synthesis and release of ANP/CDD in the blood is modulated by stretching of the atria after ‘expansion’ of the extracellular fluid volume brought about either by changes in sodium balance or after osmotic stimuli (Brenner et al., 1990; Genest and Cantin, 1988). However, ANP,/CDD secretion may also be moduiated by other factors such as norepinephrine, epinephrine, calcitotingene-related peptide, vasopressin, and endothelin (Debinski et al., 1988; Schiebinger et al., 1987; Stach et al., 1981, Yamamoto et al., 1988) Striking localization of ANP/CDD-like immunomaterial was found in the root cell bundles (Fig. 7). The specific organization of the cells of the external sulcus and especially the anchorage of the fibrils at the basement membrane of the root cell bundles and their continuity with the basilar membrane suggest that this entire system may act as a mechanical pump (Galic and Giebel, 1989; Shambaugh, 1909; Vosteen, 1961). Furthermore, secretory activity of these cells has been proposed: it has been postulated that at high sound pressure levels, the strong vibration of the basilar membrane may lead to a locally and temporarily en-

91

hanced secretion of ‘substances’ from the root cells into the cochlear duct (Galic and Giebel, 1989). Similar mechanisms may be proposed for the organ of Corti. It is particularly intriguing because these regions contain ANP/CDD-like immunoreactivity and ANP/CDD is believed to play an important role in protecting the organism from volume overloading. During the last decade several laboratories confirmed the motile activity of the outer hair cells (Brownell, 1983) after electric, osmotic and ionic stimulation. Morphological analysis reveals that the slow motility response leads to increased cell volume (Brownell et al., 19901. Recently Brownell et al. (1990) suggested that outer hair cells may possess a unique mechanism for cell volume control. Therefore the role of ANP/CDD as a candidate for this function is worth consideration. The role of ANP/CDD as neuromodulator and neurotransmitter has been suggested in both the peripheral and the central nervous system. It has recently been demonstrated that ANP/CDD is released from the hypothalamus by a depolarizing concentration of K+ through a Ca++- dependent mechanism (Takana and Inagami, 1986). Furthermore the dependence of ganglionic ANP/CDD on cholinergic innervation has been suggested (Debinski, 1988). The presence of ANP/CDD-like immunoreactive cells in the endolymphatic sac is not surprising since its secretory function was recently discovered; a function which may be associated with a reduction in the hydrostatic fluid pressure in the rest of the cochlea (Erwall et al., 1988). The nature of the secretory substances has not yet been satisfactorily explained. However, using radioactive sulphur (Etwall et al., 19891, the presence of sulphur in the substances has been detected and may also indicate that sulphur is incorporated into an amino acid in the ANP/CDD-sequence. ANP/CDD is now accepted as a new potent diuretic, natriuretic and vasorelaxant hormone which inhibits the renin-aldosteron system, antagonizes angiotensine II and suppresses vasopressine release (Brenner et al., 1990; Genest and Cantin, 1988). We suggest that by interacting with other volume-regulating hormones ANP/CDD may provide a delicate mechanism for controlling fluid and electrolyte homeostasis in the inner ear. Its proper physiological role in the inner ear, especially in the sensory and neural tissue however, remains to be elucidated.

Acknowledgements The authors wish to thank Dr. K. Rascher for critical reading of the manuscript and Mrs. 0. Bull and Mrs. R. Schwarzer for technical assistance.

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