Endocrine cells of the APUD-system in the human lung (electron microscopy characteristics)

Lungs of 4 human fetuses (11-, 13-, 22-, 28-week-old), of 1 stillborn and of 3 mature persons, operated in connection with pulmonary cancer, have been investigated. In the fetal lungs apudocytes and neuroepithelial bodies (NEB) have been revealed. The apudocytes differ from each other by structure and size of endocrine granules. In the 11-week-old fetus P1 cells with two types of granules occur most often. Among P1 cells there are several subgroups, differing in their granule dimensions. P2 apudocytes possess granules of one type with a round core and a narrow rim of cytoplasm. P3 cells are characterized with still larger granules, a very dense core and a narrow rim. In large bronchi some groups are found, consisting of two and more endocrine cells of all three types. In the lungs of the 13-week-old fetus P1 cells are defined and a new type of cells, that contain homogenous granules, characterizing by their small size. In 22 weeks of development in the intrapulmonary bronchi apudocytes with granules specific for Ec-cells are found. NEB consists of cells and islands, possessing polymorphous granules. Various types of apudocytes are defined in large bronchi of the 22-week-old fetus. In the stillborn infant apudocytes in the lung are found very seldom. In lung of the mature persons the morphology of apudocytes is unitypical. Thus, during embryogenesis and after birth there are variable types of endocrine cells and NEB.     

Morphology of the apudocytes and neuroepithelial bodies in the lungs of rats

Endocrine cells (apudocytes) and neuroepithelial bodies (NEBs) were revealed in the lungs of rats of different ages by Grimelius' argyrophilic method. Solitary apudocytes were found among the bronchial epitheliocytes, they had the oval, columnar or triangular shape. NEBs comprise groups of argyrophilic cells, in some cases the penetration of neural fibers into the bodies and their branching in the terminals are observed. Apudocytes and NEBs in 1-, 7-, and 15-day-old rats are more numerous in the epithelium of small than big bronchi. In 21- and 30-day-old and in adult animals apudocytes and NEBs are very scarce. The elements under study are likely to take part in the early postnatal development of the rat lungs.     

Expression of CFTR and Cl(-) conductances in cells of pulmonary neuroepithelial bodies

The pulmonary neuroendocrine cell system comprises solitary neuroendocrine cells and clusters of innervated cells or neuroepithelial bodies (NEBs). NEBs figure prominently during the perinatal period when they are postulated to be involved in physiological adaptation to air breathing. Previous studies have documented hyperplasia of NEBs in cystic fibrosis (CF) lungs and increased neuropeptide (bombesin) content produced by these cells, possibly secondary to chronic hypoxia related to CF lung disease. However, little is known about the role of NEBs in the pathogenesis of CF lung disease. In the present study, using a panel of cystic fibrosis transmembrane conductance regulator (CFTR)-specific antibodies and confocal microscopy in combination with RT-PCR, we demonstrate expression of CFTR message and protein in NEB cells of rabbit neonatal lungs. NEB cells expressed CFTR along with neuroendocrine markers. Confocal microscopy established apical membrane localization of the CFTR protein in NEB cells. Cl(-) conductances corresponding to functional CFTR were demonstrated in NEB cells in a fresh lung slice preparation. Our findings suggest that NEBs, and related neuroendocrine mechanisms, likely play a role in the pathogenesis of CF lung disease, including the early stages before establishment of chronic infection and chronic lung disease.     

Innervation of pulmonary neuroendocrine cells and neuroepithelial bodies in developing rabbit lung.

We investigated the development of innervation of the pulmonary neuroendocrine cell (PNEC) system composed of single cells and organoid cell clusters, neuroepithelial bodies (NEB) in rabbit fetal and neonatal lungs. To visualize the nerve fibers and their contacts with PNECs/NEBs, we used confocal microscopy and multilabel immunohistochemistry (IHC) with pan-neural marker, synaptic vesicle protein 2 (SV2), and serotonin (5-HT) as markers for PNECs/NEBs, and smooth muscle actin or cytokeratin to identify airway landmarks. The numbers and distribution of PNEC/NEB at different stages of lung development (E16, 18, 21, 26, and P2) and the density of innervation were quantified. First PNECs immunoreactive for 5-HT were identified in primitive airway epithelium at E18 as single cells or as small cell clusters with or without early nerve contacts. At E21 a significant increase in the number of PNECs with formation of early innervated NEB corpuscules was observed. The overall numbers of PNECs/NEBs and the density of mucosal, submucosal, and intercorpuscular innervation increased with progressing gestation and peaked postnatally (P2). At term, the majority of NEBs and single PNECs within airway mucosa possessed neural contacts. Such an extensive and complex innervation of the PNEC system indicates a multifunctional role in developing lung and during neonatal adaptation.     

GABAergic signaling in the pulmonary neuroepithelial body microenvironment: functional imaging in GAD67-GFP mice

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates, but has also been reported in multiple cell types outside the CNS. A GABAergic system has been proposed in neuroepithelial bodies (NEBs) in monkey lungs. Pulmonary NEBs are known as complex intraepithelial sensory airway receptors and are part of the NEB microenvironment. Aim of the present study was to unravel a GABAergic signaling system in the NEB microenvironment in mouse lungs, enabling the use of genetically modified animals for future functional studies. Immunostaining of mouse lungs revealed that glutamic acid decarboxylase 65/67 (GAD65/67), a rate-limiting enzyme in the biosynthesis of GABA, and the vesicular GABA transporter (VGAT) were exclusively expressed in NEB cells. In GAD67-green fluorescent protein (GFP) knock-in mice, all pulmonary NEBs appeared to express GFP. For confocal live cell imaging, ex vivo vibratome lung slices of GAD67-GFP mice can be directly loaded with fluorescent functional probes, e.g. a red-fluorescent calcium dye, without the necessity of time-consuming prior live visualization of NEBs. RT-PCR of the NEB microenvironment obtained by laser microdissection revealed the presence of both GABA_A and GABA_B (R1 and R2) receptors, which was confirmed by immunostaining. In conclusion, the present study not only revealed the presence of a GABAergic signaling pathway, but also the very selective expression of GFP in pulmonary NEBs in a GAD67-GFP mouse model. Different proof of concept experiments have clearly shown that adoption of the GAD67-GFP mouse model will certainly boost future functional imaging and gene expression analysis of the mouse NEB microenvironment.     

Distribution and frequency of neuro-epithelial bodies in post-natal rabbit lung: Quantitative study with monoclonal antibody against serotonin

Summary The distribution, frequency and size of neuroepithelial bodies (NEB) were studied in lungs of rabbits during different stages of development (27-day fetus, newborn, 6, 11, 21, 28 and 56 days postnatally). NEB were visualized by immunostaining with monoclonal antibody against serotonin. Detailed quantitiation of NEB was performed by use of camera lucida drawings of immunostained serial sections from the same anatomical region, i.e. the lower lobe of the left lung. The total number of NEB was counted and expressed per epithelial length of airway, surface area and volume. The size of NEB defined as surface area as well as the position of NEB in relation to the airway bifurcations was assessed in airways of different sizes. The overall number and size of NEB were found to increase during the immediate perinatal period followed by a sharp decline at 56 days of age. The number of NEB peaked at 6 days postnatally (mean 175.5 NEB/mm³ of airway epithelium) and declined significantly (3.0 NEB/mm³) at 56 days of postnatal age. The size of NEB reached its maximum at 11 days (mean surface area 659.54 m², with the largest NEB measuring 1839.98 m²). By 56 days of age, NEB became significantly smaller (mean surface area 177.29 m²) consisting of small clusters of cells situated deep within the airway epithelium. At all ages, about half of all NEB (mean 47.6%) were localized within the small peripheral airways with up to 63.9% located at airway bifurcations. These findings indicate that the functional activity of NEB may be confined predominantly to the perinatal period. The postulated functions of NEB include those of intrapulmonary hypoxia-sensitive chemoreceptors and/or endocrine-paracrine activity in the lung. Such function(s) may be important during adaptation to extrauterine life as well as for growth and development of the lung.     

Effects of unilateral vagal stimulation on intrapulmonary neuroepithelial bodies

We studied the influence of unilateral vagal stimulation on intrapulmonary neuroepithelial bodies (NEB) in rabbits. The left vagus nerve was cut and electrically stimulated for 10 min. Animals were killed and the lungs studied with fluorescence and electron microscopy. Intensity of formaldehyde-induced fluorescence, which reflects the serotonin content in NEB, was higher on the stimulated side than on the nonstimulated side (118 +/- 7 vs. 100%, n = 8, P less than 0.001). The latter difference was found to correlate with the stimulus amplitude (r = 0.9, P less than 0.05). Ultrastructurally a decrease in the number of exocytotic dense-cored vesicle (DCV) profiles at the level of the NEB basal epithelial cell membrane was found on the stimulated side (0.32 +/- 0.10 vs. 0.45 +/- 0.16 DCV/micron of basal epithelial cell membrane, n = 8, P less than 0.05). Section of the left vagus nerve without electrical stimulation affected neither the fluorescence intensity nor the number of exocytotic DCV profiles. In animals with supranodosal or infranodosal chronic vagotomy the observed effects of unilateral vagal stimulation were no longer present. We conclude that 1) vagal stimulation increases the serotonin content of NEB; 2) it decreases the number of exocytotic DCV profiles; 3) this effect depends on the amplitude of the stimulus; 4) it is obtained through efferent vagal fibers; 5) these results are the opposite of the effects seen after exposing normal NEB to acute hypoxia; and 6) these physiological experiments corroborate a vagal innervation of NEB, which may play an important role in modulating the sensitivity and reaction of NEB to various stimuli.     

The organizational characteristics of the APUD system in mammalian lungs at different stages of ontogeny]

The organization peculiarities of APUD-system in the lungs of rabbits, rats and guinea pigs has been studied. The endocrine system in the lungs of rabbits in pre- and postnatal ontogenesis is presented by the adipocytes and neuroepithelial bodies (NEB) containing a considerable number of monoamines. The number of argyrophil adipocytes and NEBs in the lungs of 21 and more day-old adult rats seem to be less than in fetuses and newborns. Monoamines are not revealed in the endocrine rat lung structures by means of the glyoxylic acid. In the lungs of guinea pigs the single argyrophil adipocytes and NEBs are determined in the gestation period.     

Neuroendocrine lung structures and tumours: immunohistochemical study by specific markers

Out of 360 lungs or lobes surgically removed, 13 non neoplastic specimens and 16 neuroendocrine (NE) tumours are investigated with immunohistochemical methods, in order to evaluate the presence of NE structures in normal and pathological human lungs. The markers used are neuron specific enolase (NSE), chromogranin (CG) and the 80 kd antigen (80 kdAg) of NE secretory granules detected by the new monoclonal Phe-5 antibody. In non-neoplastic lung specimens, clearcut immunoreactivity for all three markers appears in NE cells, neuroepithelial bodies (NEB), NE cell-hyperplasias and dysplasias. In the same specimens 4 tumourlets with analogous clearcut immunoreactivities were also observed. The NE tumours show distinct immunoreactivity for all three antisera in the 8 well differentiated cases. The 8 poorly differentiated tumours are variably immunoreactive for NSE and present low to nil staining with antisera to CG and 80 kdAg. The immunohistochemical data are interpreted according to current views about a possible relationship between NE tumours and parent normal NE lung structures.     

Quantitation of pulmonary neuroepithelial bodies in pre- and postnatal rabbits

Summary The size, density and total number of neuroepithelial bodies (NEB) in the lungs of late fetal, neonatal, and mature rabbits were determined using fluorescence microscopy. In this study lungs from 27-, 29-, 30-, and 31-day fetuses; neonates of ages 2, 7, and 30 days; and 4- and 7(+)-month-old rabbits, were used. The total number of NEB in the entire lung of rabbits from each age group was estimated based on measurements of collapsed lung volume, average NEB diameter, and NEB density (number/mm²). Average NEB diameter increased between 27 and 29 days gestation, then remained constant at 42–44 m between 29 days gestation and two days post-partum. Thereafter the diameter was significantly reduced in the 7-day group (33.7 m) and further reduced in the 4-month group (20.3 m). NEB density was initially high in 27-day fetuses (3.87/mm²), decreased significantly by 30 days gestation, increased to a high level by 2 days post-partum, then fell steadily, reaching the lowest level in the adult (0.15/mm²). This steady decrease in density was paralleled by a large increase in lung volume. The estimated total number of NEB in the lung was constant in all age groups except for a significant drop at 30 and 31 days gestation. These data indicate that the total number of NEB is maintained into adulthood; however, the density and average diameter of NEB decreases rapidly after 2 days postpartum. A sharp decrease in both total number and density observed under fluorescence microscopy at 30 and 31 days gestation suggests a change in NEB cellular activity just prior to birth.     

Neurochemical pattern of the complex innervation of neuroepithelial bodies in mouse lungs

As best characterized for rats, it is clear that pulmonary neuroepithelial bodies (NEBs) are contacted by a plethora of nerve fiber populations, suggesting that they represent an extensive group of multifunctional intraepithelial airway receptors. Because of the importance of genetically modified mice for functional studies, and the current lack of data, the main aim of the present study was to achieve a detailed analysis of the origin and neurochemical properties of nerve terminals associated with NEBs in mouse lungs. Antibodies against known selective markers for sensory and motor nerve terminals in rat lungs were used on lungs from control and vagotomized mice of two different strains, i.e., Swiss and C57-Bl6. NEB cells were visualized by antibodies against either the general neuroendocrine marker protein gene-product 9.5 (PGP9.5) or calcitonin gene-related peptide (CGRP). Thorough immunohistochemical examination of NEB cells showed that some of these NEB cells also exhibit calbindin D-28 k (CB) and vesicular acetylcholine transporter (VAChT) immunoreactivity (IR). Mouse pulmonary NEBs were found to receive intraepithelial nerve terminals of at least two different populations of myelinated vagal afferents: (1) Immunoreactive (ir) for vesicular glutamate transporters (VGLUTs) and CB; (2) expressing P2X₂ and P2X₃ ATP receptors. CGRP IR was seen in varicose vagal nerve fibers and in delicate non-vagal fibers, both in close proximity to NEBs. VAChT immunostaining showed very weak IR in the NEB-related intraepithelial vagal sensory nerve terminals. nNOS- or VIP-ir nerve terminals could be observed at the base of pulmonary NEBs. While a single NEB can be contacted by multiple nerve fiber populations, it was clear that none of the so far characterized nerve fiber populations contacts all pulmonary NEBs. The present study revealed that mouse lungs harbor several populations of nerve terminals that may selectively contact NEBs. Although at present the physiological significance of the innervation pattern of NEBs remains enigmatic, it is likely that NEBs are receptor–effector end-organs that may host complex and/or multiple functional properties in normal airways. The neurochemical information on the innervation of NEBs in mouse lungs gathered in the present study will be essential for the interpretation of upcoming functional data and for the study of transgenic mice.     

Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor

Serotonin (5-HT) type 3 receptor (5-HT(3)-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT(3)-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT(3)-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT(3)-R) and confocal microscopy localized 5-HT(3)-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT(3)-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5-150 microM) and 5-HT(3)-R agonist 2-methyl-5-HT (5-150 microM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 +/- 5.9%) by the specific 5-HT(3)-R antagonist ICS-205-930 (50 microM), whereas katanserin and p-4-iodo-N-(2-[4-(methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca(2+) and Mg(2+) in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT(3)-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.     

Vesicular glutamate transporter 2 is expressed in different nerve fibre populations that selectively contact pulmonary neuroepithelial bodies

Pulmonary neuroepithelial body (NEB) receptors in rats receive at least four different nerve fibre populations. In addition to a spinal sensory innervation that contacts NEBs at their basal side, extensive vagal nodose sensory terminals and separate nitrergic and cholinergic nerve endings protrude between NEB cells. In the present study, antibodies against the vesicular glutamate transporter 2 (VGLUT2), a transmembrane protein responsible for loading glutamate into synaptic vesicles, were used to investigate whether some of the nerve terminals contacting NEBs in rat lungs might use glutamate as a neurotransmitter. VGLUT2 immunoreactivity (IR) was detected in extensive intraepithelial arborising nerve terminals that appeared to contact most of the NEBs. Multiple immunostaining showed VGLUT2 IR in the vagal nodose and spinal sensory nerve terminals contacting NEBs, and in another, most likely sensory, intraepithelial nerve fibre population, the origin and further characteristics of which remain to be elucidated. At least part of the VGLUT2-immunoreactive nerve fibres that contact NEBs were shown to be myelinated. The expression of VGLUT2 indicates that glutamate is stored and released as a neurotransmitter in terminals of several pulmonary (sensory) nerve fibre populations that selectively relate to the complex NEB receptors. The present study strongly suggests an involvement of glutamatergic mechanisms in the peripheral transduction of sensory stimuli from the lungs, via the release of glutamate from nerve terminals, thereby modulating the activity of NEB receptor cells or the excitability of afferent nerves.     

Postnatal maturation of neuroepithelial bodies and carotid body innervation: A quantitative investigation in the rabbit

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.     

Phosphodiesterase type 5 inhibitor sildenafil citrate does not potentiate the vasodilative properties of nebivolol in rat aorta

BACKGROUND: Sildenafil citrate (SIL) is contraindicated in patients with coronary heart disease who are treated with nitric oxide (NO) donators such as organic nitrates, as it potentiates NO-mediated vasodilation. The present study investigated whether SIL also affects the vasodilatory effects of nebivolol (NEB), a selective beta1-adrenoceptor blocker with an additional, endothelium-dependent NO-liberating property, in comparison to the combination SIL/glycerol trinitrate (GTN). METHODS AND RESULTS: Experiments were performed in isolated vessel rings of rat aorta (Wistar rats, 8-12 weeks), which had been pre-contracted with phenylephrine (10(-5) M). Isometric tension was measured by a force transducer, and cumulative concentration-response curves were obtained for each drug. The rank order of vasodilatory potency as measured by the concentration needed to achieve 50% relaxation (EC50) was GTN (0.08 microM) > SIL (1.25 microM) > or = NEB (3.5 microM). In the presence of both therapeutic (1 nM) and high (1 microM) concentrations of SIL, vasodilation of GTN was potentiated as indicated by a significant increase in vasodilatory potency (EC50 GTN + low SIL: 0.019 microM, EC50 GTN + high SIL: 0.002 microM; both P < 0.01 vs. GTN). In contrast, SIL did not potentiate the vasodilatory effect of NEB (EC50 NEB + low SIL: 5.01 microM, EC50 NEB + high SIL: 3.2 microM; n.s. vs. NEB). CONCLUSIONS: These data demonstrate that SIL does not potentiate NEB-induced vasodilation in vitro. These findings indicate that the interaction between SIL and NO-donators/organic nitrates does not apply to the NO-liberating properties of NEB. Our findings suggest that SIL may safely be used in hypertensive patients treated with NEB.     

Calcitonin gene-related peptide in hamster lung and its coexistence with serotonin: a chemical and immunocytochemical study

The mammalian lung may have an important endocrine function besides being involved in gas exchange mechanisms. A number of peptide hormones have been localized to neurons and endocrine cells in the lung where they may contribute to the regulation of local pulmonary functions. We have investigated the presence of calcitonin gene-related peptide (CGRP), in the hamster lung by radioimmunoassay and by immunocytochemistry. Measurable quantities of CGRP were detected in lung tissue. Females had higher lung tissue levels of CGRP-like immunoreactivity (IR) than males. This was not reflected in an observable increase in the intensity or distribution of CGRP-like reactivity with immunocytochemistry. Distinct CGRP-like IR was recorded in clustered (NEB) and solitary (NEC) neuroendocrine cells in neonates, weanlings and adults, including all airways from trachea (NEC only) to bronchi, bronchioles, and alveolar ducts to the level of alveoli (NEC and NEB). In adult hamsters, there seemed to be fewer immunoreactive cells, although intensity was unchanged. In addition some NEB contained serotonin-like IR, and colocalization of the peptide and the amine was noted within some cells. Intra-epithelial beaded nerve fibers, subepithelial fibers, and large-caliber nerves in the hilus region and tracheal wall were also CGRP-IR, and immunoreactive nerves were occasionally found in close association with NEB at the basal pole. Positive nerve fibers were not observed in vessels within the lung, and were sparse in the adventitia of tracheal arteries.     

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring k_tr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased k_tr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.     

Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.