Inhibition of rat carotid body glomus cells TASK-like channels by acute hypoxia is enhanced by chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) chemosensory responses to acute hypoxia. In spite of that, the primary molecular target of CIH in the CB remains unknown. A key step of the hypoxic response in the CB is the chemoreceptor cell depolarization elicited by the inhibition of K⁺ channels. Thus, we tested the hypothesis that CIH potentiates the hypoxic-induced depolarization of rat CB chemoreceptor cells by enhancing the inhibition of a background K⁺ TASK-like channel. Membrane potential, single channel and macroscopic currents were recorded in the presence of TEA and 4-aminopyridine in CB chemoreceptor cells isolated from adult rats exposed to CIH. The CIH treatment did not modify the resting membrane properties but the hypoxic-evoked depolarization increased by 2-fold. In addition, the hypoxic inhibition of the TASK-like channel current was larger and faster in glomus cells from CIH-treated animals. This novel effect of CIH may contribute to explain the enhancing effect of CIH on CB oxygen chemoreception.     

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O₂ tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.     

PCNA在CLD新生鼠肺成纤维细胞中的动态表达及意义

目的探讨增殖细胞核抗原（proliferatingeellnuelearantigen,PCNA）在CLD新生鼠肺成纤维细胞中的动态表达及意义。方法足月新生SD大鼠于生后12h内分别持续吸入85%高氧和空气,于3、7、14、21天随机处死后动物,无菌条件下采集肺组织,进行LF细胞的原代培养,应用MTT法检测细胞增殖活力。免疫组化法检测PCNA蛋白表达;Real-time PCR法检测PCNAmRNA含量。结果 MTT结果显示,高氧使得LF增殖活力增强;生后3d空气组和高氧组PCNA蛋白及mRNA的表达无统计学差异。生后7d时高氧组PCNA蛋白及mRNA的表达较空气组增加（P〈0.05）;14d、21d时增加明显（P〈0.01）。结论体内高氧使LF增殖活力增加,最终导致肺间质纤维化。     

Postnatal development of spiral ganglion cells in the rat

The postnatal development of the spiral ganglion in the albino rat was studied using light and electron microscopy. The morphological characteristics distinguishing type 1 from type 2 spiral ganglion cells were defined, and the critical period for distinguishing the two types of neurons was identified. At birth, the spiral ganglion consists of a homogeneous population of small, densely packed, spherical cells that have large cytoplasmic-to-nuclear ratios. During the first postnatal week, the cells mature slowly. At this period the myelin sheath around the cell bodies generally consists of only a few layers of loose myelin. Long glial fingers extend around the cell processes and soma, particularly the filopodial extensions of the somatic membrane. Type 2 spiral ganglion cells can be distinguished at postnatal day 8. Viewed with the phase-contrast microscope these cells are smaller and have more darkly staining nuclei and more lightly staining cytoplasm than the type 1 cells. The most characteristic ultrastructural features of the type 2 neurons are the densely packed neurofilaments in the cytoplasm and lack of compact myelin around the cell soma. By day 14, spiral ganglion cells are morphologically mature, although the myelin sheath continues to thicken. The results are discussed in relation to the electrophysiological development of the auditory system and the morphological maturation of the organ of Corti.     

Changes in oxygen sensitivity of TASK in carotid body glomus cells during early postnatal development

A post-natal increase in carotid body (CB) hypoxia responsiveness occurs at the level of carotid sinus nerve activity, intracellular calcium, cell membrane depolarization and hypoxic inhibition of O(2)-sensitive background K(+) conductance. TASK-1, TASK-1/3 and TASK-3 are functionally expressed in CB glomus cells, with TASK-1/3 providing the major part of the O(2)-sensitive TASK-like background K(+) conductance. Here we report the effects of graded hypoxia on TASK-like channel activity in CB glomus cells from rats aged 0 to 1, 6 to 7 and 16 to 18 days; the time frame of postnatal CB functional maturation. TASK was active in nearly all cell-attached patches and TASK activity during normoxia did not differ across ages. Hypoxia produced a progressive decrease in channel opening frequency with graded decreases in O(2) level and also produced glomus cell depolarization, as assessed by the shift in reversal potential of TASK single channel current. Hypoxic inhibition of TASK activity was least at P0-P1 and increased with age mainly between 6-7 and 16-18 days. The O(2)-sensitive TASK activity was significantly greater in glomus cells from P16 to P18 when compared to cells from P0 to P1 day old rats. These results support the hypothesis that postnatal carotid body functional maturation is due, at least in part, to changes in the sensitivity of TASK to the hypoxic signals generated in glomus cells.     

Division of small intensely fluorescent cells in neonatal rat superior cervical ganglion is inhibited by glucocorticoids

Postnatal genesis of small, intensely fluorescent cells was studied in the rat superior cervical ganglion by combining immunocytochemistry of tyrosine hydroxylase with tritiated thymidine auto-radiography. After injection of tritiated thymidine during the first postnatal week, silver grains were observed over the nuclei of many small cells with intense staining for tyrosine hydroxylase, suggesting that SIF cells are dividing postnatally. Cell counts in ganglia of rats sacrificed 2 h after tritiated thymidine showed that the rate of SIF cell proliferation was highest during the first postnatal week with approximately 20% of SIF cells dividing and that the rate declined thereafter. Counts of labeled SIF cells at 30 days in rats injected with tritiated thymidine on days 0, 2, 4, 6, 8, 10 or 14 revealed a peak of SIF cell birthdays on day 8. In these long-survival experiments, many labeled SIF cells were present in adult superior cervical ganglions. In contrast, only one labeled principal neuron was observed in a total of 450 sections. Glucocorticoid treatment of the rats during the first postnatal week paradoxically increased the number of SIF cells, but inhibited the rate of SIF cell proliferation. Dividing SIF cells immunoreactive for both tyrosine hydroxylase and phenylethanolamineN-methyltransferase were observed in glucocorticoid-treated rats.

These observations suggest that many SIF cells are dividing during the first postnatal week. After cessation of division, these cells either remain SIF cells or die, but do not differentiate into principal neurons. Since glucocorticoids do not stimulate SIF cell proliferation, they must increase the number of SIF cells by biasing the differentiation of precursor cells in the superior cervical ganglion and/or enhancing SIF cell survival.     

Development of cardiac sensitivity to oxygen deficiency: comparative and ontogenetic aspects.

Hypoxic states of the cardiovascular system are undoubtedly associated with the most frequent diseases of modern times. They originate as a result of disproportion between the amount of oxygen supplied to the cardiac cell and the amount actually required by the cell. The degree of hypoxic injury depends not only on the intensity and duration of the hypoxic stimulus, but also on the level of cardiac tolerance to oxygen deprivation. This variable changes significantly during phylogenetic and ontogenetic development. The heart of an adult poikilotherm is significantly more resistant as compared with that of the homeotherms. Similarly, the immature homeothermic heart is more resistant than the adult, possibly as a consequence of its greater capability for anaerobic glycolysis. Tolerance of the adult myocardium to oxygen deprivation may be increased by pharmacological intervention, adaptation to chronic hypoxia, or preconditioning. Because the immature heart is significantly more dependent on transsarcolemmal calcium entry to support contraction, the pharmacological protection achieved with drugs that interfere with calcium handling is markedly altered. Developing hearts demonstrated a greater sensitivity to calcium channel antagonists; a dose that induces only a small negative inotropic effect in adult rats stops the neonatal heart completely. Adaptation to chronic hypoxia results in similarly enhanced cardiac resistance in animals exposed to hypoxia either immediately after birth or in adulthood. Moreover, decreasing tolerance to ischemia during early postnatal life is counteracted by the development of endogenous protection; preconditioning failed to improve ischemic tolerance just after birth, but it developed during the early postnatal period. Basic knowledge of the possible improvements of immature heart tolerance to oxygen deprivation may contribute to the design of therapeutic strategies for both pediatric cardiology and cardiac surgery.     

NO modulation of carotid body chemoreception in health and disease

Nitric oxide (NO), at physiological concentrations, is a tonic inhibitory modulator of carotid body (CB) chemosensory discharges. NO modulates the chemoreception process by several mechanisms, indirectly by modifying the vascular tone and oxygen delivery, and directly through the modulation of the excitability of glomus cells and petrosal neurons. In addition to the inhibitory effect, at high concentrations NO has a dual dose-dependent effect on CB chemoreception that depends on the P_O2${\text{P}}_{{\text{O}}_2}$. In hypoxic conditions, NO is primarily an inhibitory modulator of CB chemoreception, while in normoxia NO increases the chemosensory discharges. In this review, we will examine new evidence supporting the idea that NO is involved in the CB chemosensory potentiation induced by congestive heart failure (CHF) and chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea (OSA). Evidence from patients and experimental animal models indicates that CHF and OSA, as well as CIH, potentiate the carotid hypoxic chemoreflexes, contributing to enhance the sympathetic tone. Moreover, animals exposed to CIH or to pacing-induced CHF showed enhanced baseline CB discharges in normoxia and potentiated chemosensory responses to acute hypoxia. Several molecules and pathways are altered in CHF, OSA and CIH, but the available evidence suggests that a reduced NO production in the CB plays an essential role in both diseases, contributing to enhance the CB chemosensory discharges.     

Postnatal development of carotid body glomus cell response to hypoxia

This study examines developmental changes in CB glomus cell depolarization, intracellular calcium ([Ca(2+)](i)) and the magnitude of an O(2)-sensitive background ionic conductance that may play roles in the postnatal increase in oxygen sensitivity of glomus cells isolated from rats of 1-3 days and 11-14 days postnatal age. Using fura-2 and perforated patch whole cell recordings, we simultaneously measured [Ca(2+)](i) and membrane potential (E(m)) during normoxia and hypoxia. Resting E(m) in normoxia was similar at both ages. Hypoxia caused a larger E(m) depolarization and correspondingly larger [Ca(2+)](i) response in glomus cells from 11- to 14-day-old rats compared to 1-3-day-old rats. E(m) and [Ca(2+)](i) responses to 40mM K(+) were identical between the two age groups. Under normoxic conditions both age groups had similar background conductances. Under anoxic conditions (at resting membrane potential) background K(+) conductance decreased significantly more in cells from 11- to 14-day-old rats compared to cells from 1- to 3-day-old rats. Glomus cells from newborns therefore have less O(2)-sensitive background K(+) conductance. These results support the hypothesis that postnatal maturation of glomus cell O(2) sensitivity involves developmental regulation of the expression and/or O(2)-sensitivity of background ionic conductances.     

Intact and sympathectomized carotid bodies of long-term hypoxic rats: a morphometric ultrastructural study

Summary The ultrastructure of the carotid body after exposure to hypoxia (10% O₂) for one, two or three weeks was investigated morphometrically. The study was performed on rats after unilateral removal of the superior cervical ganglion. The normally occurring bimodal distribution of type I cells, representing cells with small vesicle profile diameters (SVC) and large vesicle profile diameters (LVC) respectively, changed after one week of hypoxia into a unimodal population. After one or two weeks of hypoxia the diameter range of dense-cored vesicle (DCV) profiles in type I cells was not different from that of DCV profiles in control LVC. After three weeks of hypoxia the DCV vesicle size was intermediate between those of control SVC and LVC. The volume density of DCV decreased after one week but returned to initial values after two and three weeks of hypoxia. At two or three weeks of hypoxia, however, the total cell volume was increased about 1.4 times which should reflect an increase of the total content of DCV at these times of exposure to hypoxia. An increased mean area of cell profiles indicates a hypertrophy of the type I cells, but no signs of hyperplasia could be detected. The ganglionectomy did not cause any remarkable changes compared to the intact carotid body except for a higher volume density of DCV during the early periods of hypoxia.It is inferred from the study that the increased total mass of type I cell tissue during long-term hypoxia is due to a hypertrophy of the cells. Furthermore, the type I cells can increase their storage capacity for catecholamines during hypoxia by an increase in the size and number of DCV.     

Effects of hypobaric hypoxia on histochemical fibre-type composition and myosin heavy chain isoform component in the rat soleus muscle

Histochemical fibre-type composition and myosin heavy chain isoform component in the soleus muscle were studied in normoxic rats at postnatal ages of 5, 10, 15, and 20 weeks and in rats exposed to hypobaric hypoxia (460 torr) for 5 weeks from postnatal ages of 5, 10, and 15 weeks. The increase in the percentage of type I fibres and the concomitant decrease in that of type IIa fibres in the soleus muscle of normoxic rats were observed until 15 weeks of age. On the other hand, no change in the fibre-type composition of the muscle during postnatal development was observed in hypoxic rats, irrespective of the age at which they were exposed to hypoxia. The changes in the myosin heavy chain isoform component (MHC I and MHC IIa) of the muscle during postnatal development and by hypoxia corresponded well with those in the muscle fibre-type composition. It is concluded that hypobaric hypoxia inhibits the growth-related shift of muscle fibre-types from type IIa to type I and of myosin heavy chain isoforms from MHC IIa to MHC I in the rat soleus muscle, and that there are no changes in the muscle fibre-type composition or the myosin heavy chain isoform component caused by hypoxia after the shifts in these parameters which occur during postnatal development are completed.     

Neuropeptide Y- and catecholamine-synthesizing enzymes: immunoreactivities in the rat carotid body during postnatal development

Immunocytochemical studies of the rat carotid body during postnatal development revealed neuropeptide Y (NPY), tyrosine hydroxylase (TH), and dopamine beta-hydroxylase (DBH) immunoreactivities. In adult rats (at postnatal week 10), NPY and DBH immunoreactivities were shown in a few small chief cells (cell number/section shown as mean +/- SD: NPY 3.4+/-2.6, DBH 3.2+/-2.3), in large ganglion cells, and in numerous varicose nerve fibers of the carotid body. TH immunoreactivity was found in almost all chief cells, in a few ganglion cells, and in numerous varicose nerve fibers in the carotid body. By using the double-immunostaining technique, most NPY-immunoreactive chief cells, ganglion cells, and nerve fibers exhibited DBH immunoreactivity. The NPY- and DBH-immunoreactive chief cells in the rat carotid body were numerous from birth (NPY 93.8+/-14.9, DBH 89.7+/-12.3) to postnatal week 1 (NPY 65+/-14.5, DBH 61.6+/-11.3), but decreased quickly from postnatal week 2 (NPY 6.1+/-3.5, DBH 3.6+/-2.8) onwards. A few NPY- and DBH-immunoreactive ganglion cells were found in the periphery or in the center of the rat carotid body during postnatal development. TH immunoreactivity was observed in almost all chief cells and in a few ganglion cells in all developmental stages. NPY- and DBH-immunoreactive nerve fibers were very scarce in the carotid body from birth to postnatal week 1, began to increase gradually after postnatal week 2, and reached the adult level by postnatal week 5. The present study suggests that the expression of NPY and noradrenaline in chief cells and in the nerve fibers of the rat carotid body may be regulated during postnatal development.     

Damaging impacts at the critical time periods of prenatal ontogenesis as a factor modifying the cerebral structural development and postnatal behavior reactions

Results of a study of the nature of changes in the rat neocortex, which were observed during an early postnatal period (postnatal days 1 to 10) and which were induced by a single prenatal hypoxia on the 16th or 19th embrionic day (E16, E19), are presented in the paper. Acute hypoxia, administered on E16, was shown to result in an underdevelopment of cortical layers as well in damage to cell orientation and differentiation, i.e. it disturbed the histogenic processes (proliferation, migration and differentiation), which are most active at this time period. An immunohistochemical examination of the brain made during the postnatal period after an intrauterine hypoxia suggests that damage to proliferation and differentiation occurred in glial cells. Hypoxia administered on E19, when the level of proliferation in the brain was lower, had a less pronounced damaging effect. Deviations in the neocortical structure and in the animal's behavior found during the postnatal period could be caused by the heterochromic and heteromorphous development of brain regions in the fetus.     

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.     

Carotid chemoreceptor “resetting” revisited

Carotid body (CB) chemoreceptors transduce low arterial O₂ tension into increased action potential activity on the carotid sinus nerves, which contributes to resting ventilatory drive, increased ventilatory drive in response to hypoxia, arousal responses to hypoxia during sleep, upper airway muscle activity, blood pressure control and sympathetic tone. Their sensitivity to O₂ is low in the newborn and increases during the days or weeks after birth to reach adult levels. This postnatal functional maturation of the CB O₂ response has been termed “resetting” and it occurs in every mammalian species studied to date. The O₂ environment appears to play a key role; the fetus develops in a low O₂ environment throughout gestation and initiation of CB “resetting” after birth is modulated by the large increase in arterial oxygen tension occurring at birth. Although numerous studies have reported age-related changes in various components of the O₂ transduction cascade, how the O₂ environment shapes normal CB prenatal development and postnatal “resetting” remains unknown. Viewing CB “resetting” as environment-driven (developmental) phenotypic plasticity raises important mechanistic questions that have received little attention. This review examines what is known (and not known) about mechanisms of CB functional maturation, with a focus on the role of the O₂ environment.     

Development and cytodifferentiation of peritubular myoid cells in the rat testis.

The cytodifferentiation of peritubular myoid cells was studied in developing rats from fetal day 18 through approachment of puberty. The parameters taken into consideration were 1) the presence of desmin, a component of intermediate filaments in contractile cells; 2) the expression of alkaline phosphatase, a cell surface enzyme present in no other cell type of the seminiferous tubule; 3) the expression of the smooth muscle specific isoform of alpha-actin, a marker of terminal differentiation in smooth muscle cells; 4) cell proliferation rate, evaluated in radioautography as labeling index after incorporation of 3H-thymidine in short-term organ culture; and 5) cytoarchitectural changes detected with scanning electron microscopy. By means of immunofluorescence and cytochemistry it was observed that the three markers are expressed early during life, long before the onset of the first spermatogenic wave; in particular desmin is already present in fetal samples and alkaline phosphatase activity appears a few days after birth, whereas alpha-smooth muscle isoactin is first detected around birth. As for myoid cell replication, the high prenatal labeling index was found to drop soon after birth and to further slow down during the first month of postnatal life, suggesting that myoid cell proliferation is not a major factor in peritubular expansion. SEM examination of developing peritubulum has shown that, when approaching puberty, the myoid cell undergoes a dramatic change in cytoarchitecture, consisting in extreme flattening and cytoplasmic expansion resulting in an apparent increase in peritubular surface.     

Cell formation in the human hippocampal formation from mid-gestation to the late postnatal period

In the present study cell formation was studied in the human hippocampal formation from the 24th gestational week until the end of the first postnatal year. Proliferating cells were detected with the monoclonal antibody MIB-1.The cytoarchitectonic layers of Ammon's horn are formed before the 24th gestational week. In harmony with this observation, cell proliferation in the hippocampal ventricular zone is minimal after the 24th week. In addition, local cell multiplication in Ammon's horn is occasional and the proliferating cells are glial or endothelial cells. In contrast, cell formation continues in the hilar region of the dentate gyrus even after birth. Immature cells accumulate in the hilus, and at the border between the hilus and the granule cell layer throughout the first eight postnatal months. The subgranular zone of the dentate gyrus becomes a cell sparse area at about the 11th postnatal month, indicating that immature cells from the hilus have already migrated to the granule cell layer and differentiated into granule cells. There is an increase in glial cell proliferation both in Ammon's horn and the dentate gyrus at the 11.5th postnatal month suggesting the onset of myelination by the end of the first year.Our findings indicate that most pyramidal neurons of Ammon's horn are generated in the first half of pregnancy and no pyramidal neurons are formed after the 24th gestational week. In contrast, granule cells of the dentate gyrus proliferate in a decreasing rate during the second half of pregnancy and after birth. Proliferating neuronal precursors occur in a low percentage in the dentate gyrus of 3-, 5- and 11.5-month-old children.     

Neonatal neuronal loss in rat superior cervical ganglia: retrograde effects on developing preganglionic axons and Schwann cells

Summary Beginning prenatally and during the first week after birth, there is normally a loss of axons in rat cervical sympathetic trunk. To test the hypothesis that this spontaneous axonal loss represents a natural process whereby an excessive number of immature preganglionic axons in the cervical sympathetic trunk adapts to the neuronal population in the superior cervical ganglion, the number of nerve cells in the superior cervical ganglion was reduced in newborn rats by administration of nerve growth factor antiserum, 6-hydroxy-dopamine or postganglionic axotomy. Quantitative ultrastructural studies of these animals at later stages of development revealed that, with each method, the number of preganglionic axons and Schwann cells was reduced to nearly one-third of normal. These findings indicate that the superior cervical ganglion plays an important role in the development of the cervical sympathetic trunk. Removal of ganglionic cells causes a retrograde loss of preganglionic fibres. This process probably represents an exaggeration of the normal mechanism for elimination of redundant axons. Because the changes in axonal numbers are associated with similar reductions in the number of Schwann cells, it can also be concluded that postnatal Schwann cell proliferation is influenced by axonal populations.     

Postnatal development of carotid body glomus cell O2 sensitivity

In mammals, the main sensors of arterial oxygen level are the carotid chemoreceptors, which exhibit low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life. This postnatal increase in hypoxia sensitivity of the arterial chemoreceptors, termed "resetting", remains poorly understood. In the carotid body, hypoxia is transduced by glomus cells, which are secretory sensory neurons that respond to hypoxia at higher P(O2) levels than non-chemoreceptor cell types. Maturation or resetting of carotid body O2 sensitivity potentially involves numerous aspects of the O2 transduction cascade at the glomus cell level, including glomus cell neurotransmitter secretion, neuromodulator function, neurotransmitter receptor expression, glomus cell depolarization in response to hypoxia, [Ca2+]i responses to hypoxia, K+ and Ca2+ channel O2 sensitivity and K+ channel expression. However, although progress has been made in the understanding of carotid body development, the precise mechanisms underlying postnatal maturation of these numerous aspects of chemotransduction remain obscure.