Immunohistochemical localization of calbindin D28-k,parvalbumin, and calretinin in the cerebellar cortex of the circling mouse

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.     

Postnatal development of parvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat

Parvalbumin and calbindin D28k immunoreactivities were examined in the neocortex of the rat during postnatal development. Parvalbumin-immunoreactive nonpyramidal neurons first appear in layer V and later in layers VI and IV, and then in II and III. Immunoreactive terminals forming baskets surrounding unlabelled somata appear about 2 days later. The first parvalbumin-immunoreactive neurons appear in the retrosplenial and cingulate cortices, and the rostral region of the primary somatosensory cortex at postnatal days 8 or 9 (P8–P9). These regions are followed by the primary visual, primary auditory and motor cortices at P11. Parvalbumin immunoreactivity appears last in the secondary areas of the sensory regions and association cortices. Adult patterns are reached at the end of the 3rd week. Calbindin D28K-immunoreactive nonpyramidal neurons are found at birth in all cortical layers excepting the molecular layer. The intensity of the immunoreaction increases during the first 8 or 11 days of postnatal life, first in the inner and later in the upper cortical layers, following, therefore, an inside-out gradient. Heavily-labelled calbindin D28K-immunoreactive nonpyramidal cells dramatically decrease in number from P11 to P15 due mainly to a decrease of the multipolar subtypes. This suggests that two populations of calbindin D28k-immunoreactive nonpyramidal neurons are produced in the neocortex during postnatal development: one population of neurons transitorily expresses calbindin D28k immunoreactivity; the other population is composed of neurons that are permanently calbindin D28k immunoreactive. In addition to heavily labelled nonpyramidal cells, a band of weakly labelled pyramid-like neurons progressively appears in layers II and III throughout the cerebral cortex, beginning in layer IV in the somatosensory cortex by the end of the 2st week. Adult patterns are reached at the end of the 3rd week. These results indicate that parvalbumin and calbindin D28k immunoreactivities in the cerebral neocortx follow different characteristic patterns during postnatal development. The appearance of parvalbumin immunoreactivity correlates with the appearance of the related functional activity in the different cortical regions, and, probably, with the appearance of inhibitory activity in the neocortex. On the other hand, the early appearance of calbindin D28k immunoreactivity in the neocortex may be related to the early appearance of calbindin immunoreactivity in many other brain regions, and suggests another, as yet unknown, role for this calcium-binding protein during development of the cerebral cortex.     

Ontogeny of the calcium binding protein calbindin D-28k in the rat nervous system

Summary Calbindin D-28k immunoreactivity appeared at embryonal day 14 (E14) in the central nervous system as well as in the sensory organs and at E15 in the peripheral nervous system of the rat. At E14 the infundibular process of the diencephalon, cells of the posterior hypothalamus and of the dorsal thalamus were the only structures strongly immunostained in the brain, whereas neurons of the basal plate of the spinal cord, medulla oblongata and of the out-ermost layer of the cerebral cortex were only faintly labeled. Calbindin positive cerebellar Purkinje cells could be discerned at E15 together with a few cells in the hippocampus and in ganglia of the cranial nerves. At E19 various mesencephalic and metencephalic structures, spinal ganglion cells and basal ganglia displayed calbindin immunoreactive cells. The adult pattern of calbindin immunoreactivity (Garcia Segura et al. 1984) was reached before birth in most brain regions. In general, cells which displayed calbindin during brain development were also calbindin positive in the adult animal. Exceptions to this rule were cells of the deep nuclei of the cerebellum and non-neuronal cells which transiently expressed calbindin during development. Calbindin appeared in a given brain region almost invariably 1 or 2 days after the cessation of cell division and the beginning of neuronal migration and extension of neuronal processes. The calcium binding protein calbindin might influence these Ca²⁺-dependent processes.Abbreviations A Axon - ac anterior commissure - Acq Aqueductus cerebri - AH Adenohypophysis - AMY Amygdala - aV anterior vermis - BG Basal Ganglia - BO Bulbus olfactorius - BPG basal pontine grey - C Cortex - CA Crista ampullaris - cer Cerebellum - CO otic cyst - CP choroid plexus - CPT Caudatoputamen - DCN deep cerebellar nuclei - DT dorsal thalamus - E foregut epithelium - ec external capsule - eml external medullary lamina - ENP entopeduncular nucleus - EP Ependym - ET Epithalamus - EY eye - F Fimbria - fo Fornix - fr Fasciculus retroflexus - GP Globus pallidus - gr granular laver of the cerebellum - Gsp ganglion spirale cochlea - HI Hippocampus - HYP Hypothalamus - I hippocampal interneurons - ic internal capsule - IE inner ear - IH inner hair cells of the cochlea - IO inferior olive - IS internal sulcus cells of the cochlea - LD laterodorsal thalamus - LV lateral ventricle - MB mamillary body - MH medial habenular nucleus - mol molecular layer of the cerebellum - mt mamillo-thalamic tract - mtt mamillo-tegmental tract - N nose - n olfactory nerve - NBM Nucleus basalis of Meynert - NE nose epithelium - NH Neurohypophysis - NRT Nucleus reticularis tegmenti pontis - ON olfactory nuclei - OR optic recess - PC posterior commissure - PG Epiphysis - PI inferior cerebellar peduncle - PS superior cerebellar peduncle - PU Purkinje cell - PV paraventricular hypothalamic nucleus - pV posterior vermis - PVP paraventricular thalamic nucleus - PY pyramidal cells of the hippocampus - RA Raphe nuclei - RE Nucleus reuniens - RH Nucleus rhomboideus - RN reticular nucleus - RP Rathke's pouch - sc spinal cord - SG substantia gelatinosa Rolandi - slm Stratum-lacunosum molecular - sm Stria medullaris - SN Substantia nigra - SO supraoptic nucleus - Sol Nucleus of the solitary tract - Tect Tectum - Teg Tegmentum - TG tegmental nucleus of Gudden - Tg tongue - TO tuberculum olfactorium - VT ventral thalamus - WH white matter - ZI Zona incerta - 3 third ventricle - 4 fourth ventricle - II _N Nervus opticus - Vg Trigeminal ganglion - Vsp spinal trigeminal nucleus - VIIG Ganglion geniculi - VIIIG Ganglion vestibulare - IXGI interior ganglion of IX - IXGS superior ganglion of IX - XGI inferior ganglion of X Submitted by S.E. as her doctoral thesis at the medical faculty of the University of Zürich     

Prenatal acoustic stimulation influences neuronal size and the expression of calcium-binding proteins (calbindin D-28K and parvalbumin) in chick hippocampus

Prenatal auditory enrichment by species-specific sounds and sitar music enhances the expression of immediate early genes, synaptic proteins and calcium binding proteins (CaBPs) as well as modifies the structural components of the brainstem auditory nuclei and auditory imprinting area in chicks. There is also facilitation of postnatal auditory preference of the chicks to maternal calls following both types of sound stimulation indicating prenatal perceptual learning. To examine whether the sound enrichment protocol also affects the areas related to learning and memory, we assessed morphological changes in the hippocampus at post-hatch day 1 of control and prenatally sound-stimulated chicks. Additionally, the proportions of neurons containing calbindin D-28K and parvalbumin immunoreactivity as well as their protein levels were determined. Fertilized eggs of domestic chick were incubated under normal conditions of temperature, humidity, forced draft of air as well as light and dark (12:12 h) photoperiods. They were exposed to patterned sounds of species-specific and sitar music at 65 dB for 15 min per hour over a day/night cycle from day 10 of incubation till hatching. The hippocampal volume, neuronal nuclear size and total number of neurons showed a significant increase in the music-stimulated group as compared to the species-specific sound-stimulated and control groups. However, in both the auditory-stimulated groups the protein levels of calbindin and parvalbumin as well as the percentage of the immunopositive neurons were increased. The enhanced proportion of CaBPs in the sound-enriched groups suggests greater Ca²⁺ influx, which may influence long-term potentiation and short-term memory.     

Ontogeny of two calcium-binding proteins (calbindin D-28K and parvalbumin) in the human inferior olivary complex and their distribution in the adults

The inferior olivary complex (IOC) is a prominent nuclear relay system of the medulla oblongata. Anatomically, it is connected to the cerebellum for coordination of motor activities. Calbindin D-28K (CALB) and parvalbumin (PV) are cytosolic calcium-binding proteins (CBP) that play a role in Ca²⁺ homeostasis. We examined their ontogeny and distribution in the fetal, postnatal and adult human IOC by immunohistochemistry. At 11–12 weeks of gestation (wg), calbindin immunoreactivity was present in the principal olive and the medial accessory olive, it was absent in the dorsal olive. Parvalbumin immunoreactivity developed at 16–17 wg in the ventral lamella and the lateral bulge of the principal olive only. Calbindin expression gradually increased from 20 to 37 wg, whilst by contrast, parvalbumin expression was moderate. By 37 wg, all three IOC subnuclei were immunopositive for both proteins. In a 3-month-old infant, parvalbumin was intensely developed in the olivary axons. In the adults (40- to 59-year-old), calbindin was distributed in most neurons, and olivocerebellar fibres, whereas parvalbumin was present in some neurons and few fibres. Parvalbumin expressed till 51 years, and disappeared by 59 years of age. Calbindin immunoreactivity in the olivary axons was declined at 70 years of age. The data suggest a differential distribution and requirement of these proteins in the human IOC maturation. It may be that the IOC utilizes mainly calbindin for Ca²⁺ buffering. The loss of parvalbumin with ageing might influence the excitability of the spared IOC neurons.     

Postnatal development of calcium-binding proteins immunoreactivity (parvalbumin, calbindin, calretinin) in the human entorhinal cortex

The entorhinal cortex is an essential component in the organization of the human hippocampal formation related to cortical activity. It transfers, neocortical information (ultimately distributed to the dentate gyrus and hippocampus) and receives most of the hippocampal output directed to neocortex. At birth, the human entorhinal cortex presents similar layer organization as in adults, although layer II (cell islands) and upper layer III have a protracted maturation. The presence of interneurons expressing calcium-binding proteins (parvalbumin, calbindin–D28K (calbindin) and calretinin) is well documented in the adult human entorhinal cortex. In many of them the calcium binding is co-localized with GABA. Parvalbumin-immunoreactive cells and fibers were virtually absent at birth, their presence increasing gradually in deep layer III, mostly in the lateral and caudal portions of the entorhinal cortex from the 5th month onwards. Calbindin immunoreactive cells and fibers were present at birth, mainly in layers II and upper III; mostly at rostral and lateral portions of the entorhinal cortex, increasing in number and extending to deep layers from the 5th month onwards. Calretinin immunoreactivity was present at birth, homogeneously distributed over layers I, II and upper V, throughout the entorhinal cortex. A substantial increase in the number of calretinin neurons in layer V was observed at the 5th month. The postnatal development of parvalbumin, calbindin and calretinin may have an important role in the functional maturation of the entorhinal cortex through the control of hippocampal, cortical and subcortical information.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.     

Calcium-binding proteins in carcinoma,neuroblastoma and glioma cell lines

Summary Antisera against the Ca²⁺-binding proteins parvalbumin, calbindin D-28K, and the S-100 proteins were used to study the distribution of their target proteins in selected human carcinoma (LICR-HN6;Caco-2), mouse neuroblastoma (clone NB-2a), and rat glioma cell lines (clone C-6). Pronounced staining with anti-parvalbumin was observed in the cytosol of all cells as well as in some nuclei, in particular, mitotic nuclei were highly immuno-reactive. Applying light and immune-electron microscopy (colloidal gold labelling) the parvalbumin-fluorescence was associated with filaments in the LICR-HN6 cells. However, this immunoreactivity was not a result of the presence of parvalbumin itself - as shown by biochemical analyses (HPLC, 2D-PAGE) - but was due to the presence of a Ca²⁺-binding and tumour-associated protein with similar biochemical and immunological properties. S-100 proteins were present in all tumour cell lines but their intracellular distribution was different from calbindin D-28K. Calbindin-immunoreactivity was found on the membranes of the carcinoma cell lines whereas neuroblastoma and glioma cells remained unlabelled. It is suggested that these proteins might be involved in the modulation of the enhanced stimulation of Ca²⁺-dependent processes occurring in tumour cells.     

The distribution of calbindin, calretinin and parvalbumin immunoreactivity in the human thalamus

Calcium-binding proteins show a heterogeneous distribution in the mammalian central nervous system and are useful markers for identifying neuronal populations. The distribution of the three major calcium-binding proteins - calbindin-D28k (calbindin), calretinin and parvalbumin - has been investigated in eight neurologically normal human thalami using standard immunohistochemical techniques. Most thalamic nuclei show immunoreactive cell bodies for at least two of the three calcium-binding proteins; the only nucleus showing immunoreactivity for one calcium-binding protein is the centre médian nucleus (CM) which is parvalbumin-positive. Overall, the calcium-binding proteins show a complementary staining pattern in the human thalamus. In general terms, the highest density of parvalbumin staining is in the component nuclei of the ventral nuclear group (i.e. in the ventral anterior, ventral lateral and ventral posterior nuclear complexes) and in the medial and lateral geniculate nuclear groups. Moderate densities of parvalbumin staining are also present in regions of the mediodorsal nucleus (MD). By contrast, calbindin and calretinin immunoreactivity both show a similar distribution of dense staining in the thalamus which appears to complement the pattern of intense parvalbumin staining. That is, calbindin and calretinin staining is most dense in the rostral intralaminar nuclear group and in the patchy regions of the MD which show very low levels of parvalbumin staining. However, calbindin and calretinin also show low levels of staining in the ventral nuclear complex and in the medial and lateral geniculate bodies which overlaps with the intense parvalbumin staining in these regions. These results show that the calcium-binding proteins are heterogeneously distributed in a complementary fashion within the nuclei of the human thalamus. They provide further support for the concept recently proposed by Jones (Jones, E.G., 1998. Viewpoint: the core and matrix of thalamic organization. Neuroscience 85, 331-345) that the primate thalamus comprises of a matrix of calbindin immunoreactive cells and a superimposed core of parvalbumin immunoreactive cells which may have differential patterns of cortical projections.     

钙激活性氯离子通道与高肺血流性肺动脉高压

Pulmonary hypertension induced by high pulmonary blood flow involves a variety of complex mechanisms, including endothelial damage, pulmonary artery smooth muscle relaxation-contraction disorder and vascular remodeling. Besides, the factor of ion channels in pulmonary artery smooth muscle cells is also highly correlated to vasoconstriction. In recent years, many studies have shown that activation of Ca²⁺-activated Cl^- channels is responsible for the membrane depolarization of pulmonary artery smooth muscle cells, and plays an important role in the regulation of vascular tone and vasoconstriction. This article reviews the biophysical and pharmacological characteristics of Ca²⁺-activated Cl^- channels as well as the influence of Ca²⁺-activated Cl^- channels in high pulmonary blood flow-induced pulmonary hypertension.     

The immunocytochemical distribution of calbindin-D28k and parvalbumin in identified neurons of the pulvinar-lateralis posterior complex of the cat

The calbindin-D28k and parvalbumin immunoreactivities of the neurons of the pulvinar-lateral posterior complex (Pul-LP) were studied in the cat. The neurons of the Pul-LP projecting to the cerebral cortex were identified by a retrogradely transported tracer injected in the suprasylvian gyrus. Two populations of cells were found, a calbindin-D28k-immunoreactive, large-diameter population and a parvalbumin-immunoreactive, small-diameter group. The two kinds of cells are closely intermingled. The former includes the neurons retrogradely marked, and therefore projecting to the suprasylvian gyrus. The latter includes neurons which were not retrogradely marked, and therefore presumably intrinsic elements.     

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

The perforant path in the seizure sensitive gerbil contains the Ca2+-binding protein parvalbumin

Summary The Mongolian gerbil (Meriones unguiculatus) is used as a model in epilepsy studies. Structural abnormalities in the hippocampus and in its GABAergic system have been correlated with this affliction. A reliable marker of a subpopulation of GABAergic neurons is the Ca²⁺ binding protein parvalbumin (PV). Here we show that, whereas PV is present in the same population of hippocampal interneurons in gerbil as described in the rat, in the gerbil, PV-immunoreactivity is also found in the outer molecular layer of the hippocampus. Ultrastructural analysis revealed that it is located there in axospinous boutons with asymmetric synaptic junctions, i.e. the terminals of the entorhinal perforant path. Upon ablation of the intensely PV-immunoreactive entorhinal cortex, PV-staining is completely absent in its hippocampal termination zones. Thus, in gerbil hippocampus (but not in the rat, mouse, cat and man) PV is contained in a presumably excitatory projection. This outstanding feature of the limbic system of the gerbil implies different functional properties related to Ca²⁺ mediated processes, and could be of relevance for the seizure sensitivity of this animal species.     

Localization of parvalbumin mRNA in rat brain by in situ hybridization histochemistry

Summary Parvalbumin mRNA was localized in rat brain by in situ hybridization using a ³⁵S labelled rat parvalbumin cDNA and a synthetic oligodeoxyribonucleotide (corresponding to base sequences 140 to 183 of rat parvalbumin cDNA). Strongest hybridization signals were detected in the Purkinje cells of the cerebellum and in neurones of the reticular nucleus of the thalamus. Signal was also detected in the cerebral cortex, hippocampus, basal ganglia and brain stem in agreement with the distribution of parvalbumin immunoreactivity.     

Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex

Chandelier cells are cortical GABAergic interneurons with a unique synaptic specificity enabling them to exert a strong inhibitory influence on pyramidal cells. By using immunocytochemistry for the calcium-binding protein calbindin D-28k in the human temporal neocortex, we have found numerous immunoreactive processes that were identified as chandelier cell axon terminals. This was a striking find since in previous immunocytochemical studies of the primate neocortex, chandelier cell axon terminals had been shown to be immunoreactive for another calcium-binding protein, parvalbumin, and colocalization studies indicate that parvalbumin and calbindin are present in almost completely separate neuronal populations. Here, we present double-label immunofluorescence experiments showing that parvalbumin and calbindin immunoreactivities are colocalized in certain neurons that include a subpopulation of chandelier cells whose cell bodies are located mainly in layers V and VI of the human temporal neocortex. The results suggest a selective laminar distribution of neurochemical subtypes of chandelier cells which is a peculiar feature of the organization of the human neocortex.     

Developmental changes of the distribution of binding sites for organic Ca2+-channel blockers in cat visual cortex

Summary There are indications that during a critical period of visual cortex development Ca²⁺-fluxes from extra- to intracellular compartments serve as a trigger signal for experience-dependent changes of neuronal response properties. In this study we investigate the possibility of a relation between the time course of the critical period and age-dependent changes in the density and topographical distribution of Ca²⁺-channels. As a marker for the latter we used Ca²⁺-channel blockers of the 1,4-Dihydropyridine (1,4-DHP) class since these are supposed to bind to voltage-dependent Ca²⁺-channels. We used the tritiated 1,4-DHP derivative ³H-PN 200 110 for autoradiographic determination of 1,4-DHP binding sites in the visual cortex of adult cats and kittens ranging in age from two to ten weeks. The binding of ³H-PN 200 110 to slide-mounted tissue sections was saturable and of high affinity. The overall density of specific ³H-PN 200 110 binding sites decreased during development and their laminar distribution underwent marked changes: in young kittens specific binding was accentuated in lower layer IV, whereas in adult cats the supragranular layers were most intensely labeled. Dark rearing did not affect these developmental changes of ³H-PN 200 110 binding sites. The time course of the reduction of 1,4-DHP binding sites correlates well with that of the age-dependent decrease of the susceptibility to experience-dependent modifications. We consider this result as compatible with the hypothesis that use-dependent modifications of the response properties of cortical neurons involve changes in the Ca²⁺-fluxes from extra- to intracellular compartments.     

Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line

We have proposed recently that a pertussistoxin-insensitive Ca²⁺ influx stimulated by Y₂-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca²⁺ and independent of internal Ca²⁺ stores. We describe here the actions of NPY in these same cells, using the activity of Ca²⁺-activated K⁺ channels as an indicator of [Ca²⁺]_i. The elementary slope conductance of these channels was 110±3 pS (with an asymmetrical K⁺gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca²⁺, increased the channel open probability. ATP applied in the absence of external Ca²⁺ caused rises both in channel open probability and [Ca²⁺]_i. Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca²⁺ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5-triphosphate (GTP) by (guanosine 5-O-(2-thiodiphosphate) (GDP[S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y₂-type receptors to Ca²⁺ influx in CHP-234 cells.     

Differential distribution of two Ca2+-dependent and -independent K+ channels throughout receptive and basolateral membranes of bullfrog taste cells

We could identify two types of K⁺ channels, of 80 and 40 pS conductance, respectively, in the bullfrog taste cell membrane using excised and cell-attached configurations of the patch-clamp technique. The taste cell membrane could be divided into four membrane parts — receptive area, apical process, cell body and proximal process. The 80-pS K⁺ channels were dependent on voltage and Ca²⁺ and were located exclusively on the receptive membrane and the apical process membrane. The 40-pS K⁺ channels were independent of voltage and Ca²⁺. The open probability of 40-pS K⁺ channels was decreased by the simultaneous presence of cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), and the suppressive effect was antagonized by protein kinase inhibitor (PKI). Although 40-pS K⁺ channels were found in a high density on the receptive and apical process membranes, the channels also were present in the other two parts of the taste cell membrane. These results suggest that the two different types of K⁺ channel in the bullfrog taste cells may play different roles in gustatory transduction.