Biomedical subjects

G H Hansen

Publications and source records attributed to G H Hansen.

At least 19 recordsLinked to original sources

PubMed2004

Intestinal tissue transglutaminase in coeliac disease of children and adults: ultrastructural localization and variation in expression.

BACKGROUND: Tissue transglutaminase is the main antigen for the anti-endomysial antibodies used for diagnosis of coeliac disease and can with some specificity in vitro deamidate gliadins generating potent epitopes. The intestinal levels and the ultrastructural localization of tissue transglutaminase in normal and affected persons were investigated to provide further information on its role in this disease. Intestinal biopsies were taken from normal and coeliac children and adults. METHODS: The level of transglutaminase was analysed by means of a quantitative enzymatic assay and its ultrastructural localization by immunogold electronmicroscopy using a monoclonal antibody against tissue transglutaminase. RESULTS: In relation to normal individuals, the enzymatic activity of tissue transglutaminase in adult coeliac patients was increased. The enzyme was found in the enterocytes and in increased amount just beneath the enterocytes, where cytosolic and nuclear labelling of distinct elongated cells was seen in addition to extracellular labelling close to collagen fibrils. In children, the enzymatic activity and the immunogold labelling could not be shown to be related to disease. In all cases the enzyme activity was EDTA-sensitive. CONCLUSIONS: The increased amount of tissue transglutaminase activity in coeliac adults was shown to be due to the appearance of the enzyme in enterocytes and increased expression in the lamina propria. No evidence was found to support the idea of a changed localization or changed amounts as primary elements in coeliac disease pathogenesis, nor for the involvement of non-calcium dependent microbial transglutaminases.

Adolescent↗

PubMed2003

Scavenger receptor class B type I (SR-BI) in pig enterocytes: trafficking from the brush border to lipid droplets during fat absorption.

BACKGROUND: Scavenger receptor class B type I (SR-BI) is known to mediate cellular uptake of cholesterol from high density lipoprotein particles and is particularly abundant in liver and steroidogenic tissues. In addition, SR-BI expression in the enterocyte brush border has also been reported but its role in the small intestine remains unclear. AIM AND METHODS: To gain insight into the possible function of pig SR-BI during uptake of dietary fat, its localisation in enterocytes was studied in the fasting state and during fat absorption by immunogold electron microscopy and subcellular fractionation. RESULTS: In the fasting state, SR-BI was mainly localised in the microvillar membrane and in apical invaginations/pits between adjacent microvilli. In addition, a subapical compartment and small cytoplasmic lipid droplets were distinctly labelled. During lipid absorption, the receptor was found in clathrin positive apical coated pits and vesicles. In addition, cytoplasmic lipid droplets that greatly increased in size and number were strongly labelled by the SR-BI antibody whereas apolipoprotein A-1 positive chylomicrons were largely devoid of the receptor. CONCLUSION: During absorption of dietary fat, SR-BI is endocytosed from the enterocyte brush border and accumulates in cytoplasmic lipid droplets. Internalisation of the receptor occurs mainly by clathrin coated pits rather than by a caveolae/lipid raft based mechanism.

Animals↗

PubMed2001

Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes.

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < or = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < or = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed >70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.

Animals↗

PubMed2001

Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains.

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and 'rosettes' made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.

Blotting, Western↗

PubMed2000

Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking.

Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical membrane trafficking in enterocytes. Cultured mucosal explants of pig small intestine were treated for 2 h with the cholesterol sequestering agent methyl-beta-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. The treatment reduced the cholesterol content >50%. Morphologically, the Golgi complex/trans-Golgi network was partially transformed into numerous 100-200 nm vesicles. By immunogold electron microscopy, aminopeptidase N was localized in these Golgi-derived vesicles as well as at the basolateral cell surface, indicating a partial missorting. Biochemically, the rates of the Golgi-associated complex glycosylation and association with rafts of newly synthesized aminopeptidase N were reduced, and less of the enzyme had reached the brush border membrane after 2 h of labeling. In contrast, the basolateral Na(+)/K(+)-ATPase was neither missorted nor raft-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking.

Animals↗

PubMed1999

Transcytosis of immunoglobulin A in the mouse enterocyte occurs through glycolipid raft- and rab17-containing compartments.

BACKGROUND & AIMS: Glycolipid "rafts" have been shown to play a role in apical membrane trafficking in the enterocyte. The present study characterized the membrane compartments of the enterocyte involved in transepithelial transport of small intestinal immunoglobulin A (IgA). METHODS: Immunogold electron microscopy and radioactive labeling of mouse small intestinal explants were performed. RESULTS: IgA and the polymeric immunoglobulin receptor/secretory component were present in a raft compartment. Raft association occurred posttranslationally within 30 minutes, preceding secretion into the culture medium. IgA labeling was seen primarily in enterocytes along the basolateral plasma membrane and over endosomes and small vesicles in the basolateral and apical regions of the cytoplasm. IgA and a brush border enzyme, aminopeptidase N, were colocalized in apical endosomes and small vesicles and were also frequently seen associated with the same vesicular profiles of glycolipid rafts. Colocalization of IgA and rab17, a small guanosine triphosphatase involved in transcytosis, was seen mainly along the basolateral plasma membrane and over basolateral endosomes and vesicles, but also in the apical region of the cytoplasm. CONCLUSIONS: IgA is transcytosed through a raft-containing compartment, most likely the apical endosomes. Our data also support the notion that rab17 is involved in transcytotic membrane traffic.

Amino Acid Sequence↗

PubMed1998

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment.

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.

Ammonium Chloride↗

PubMed1998

Effects of gamma-aminobutyric acid (GABA) on synaptogenesis and synaptic function.

The correct establishment and function of synapses depend on a variety of factors, such as guidance of pre- and postsynaptic neurons as well as receptor development and localization. gamma-Aminobutyric acid (GABA) has a pronounced effect on these events and elicits differentiation of neurons; that is, GABA acts as a trophic signal. Accordingly, activating preexisting GABA receptors, a trophic GABA signal enhances the growth rate of neuronal processes, facilitates synapse formation, and promotes synthesis of specific proteins. Transcription and de novo synthesis are initiated by the GABA signal, but the intracellular link between GABA receptor activation and DNA transcription is largely unknown. GABA also controls the induction and development of functionally and pharmacologically different GABAA receptor subtypes. The induced receptors are likely to be inserted only into the synaptic membrane domain. However, this ability to target the induced GABAA receptors is probably coupled to the maturation of neurons and not to the action of GABA per se. The induced GABAA receptors apparently mediate a pronounced inhibition of neurotransmitter release, whereas other subtypes of GABAA receptors may be modulatory rather than inhibitory.

Animals↗

PubMed1997

Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors.

Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the alpha6 GABA(A) receptor subunit. Membranes prepared from these cultures were photolabeled with the imidazobenzodiazepine [3H]Ro15-4513. In THIP-treated cultures at 4 days in vitro (DIV), photolabeled [3H]Ro15-4513 binding in membranes was significantly increased for both the 51 kilodalton, kDa, (alpha1 subunit) and 56-kDa (alpha6 subunit) radioactive peaks in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [3H]Ro15-4513 in the 51-kDa peak; however, no significant change in [3H]Ro15-4513 binding was observed for the 56-kDa polypeptide. Immunolabeling of the alpha6 subunit using silver-enhanced, immuno-gold staining of granule cells showed a significant effect with THIP treatment only at 4 DIV and not at 8 DIV. Examination by light microscopy demonstrated that the major effect of THIP was to increase alpha6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in alpha6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest that THIP has a trophic effect on alpha6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABA(A) agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABA(A) receptor subunits in the developing cerebellum.

Animals↗

PubMed1997

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity.

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms of the glycoprotein aminopeptidase N can be synthesized and the effects of altered glycosylation can be studied. It is demonstrated that aminopeptidase N carries "mucin-type" O-glycans and that this is predominantly located in the stalk, which connects the catalytic headgroup to the membrane anchor. Normally glycosylated aminopeptidase N is present in the plasma membrane of the ldl(D) cells. This is also the case for the non-O-glycosylated and defectively N-glycosylated forms. This is in line with the finding that the intracellular transport APN is unaffected by the absence of O-glycosylation or by changes in N-glycosylation as the various glycosylated forms of aminopeptidase N are normally converted from the high-mannose form to the complex glycosylated form. Enzymatic activity is not influenced by the changes in glycosylation.

Animals↗

PubMed1995

Localization and biosynthesis of aminopeptidase N in pig fetal small intestine.

BACKGROUND & AIMS: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS: This study was performed using histochemistry and immunoelectron microscopy and [35S]methionine labeling of cultured mucosal explants. RESULTS: Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg(2+)-precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing in the apical endocytic structures. CONCLUSIONS: In comparison with the adult enzyme, fetal aminopeptidase N has a more widespread subcellular distribution with substantial amounts present in apical endocytic compartments characteristic of the fetal enterocyte.

Animals↗

PubMed1995

Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist.

Quantitative analysis of the density of alpha 1 and beta 2/3 GABAA receptor subunits was performed at the electron microscope level after indirect pre-embedding immunogold labeling with subunit-specific antibodies of rat cerebellar granule cell cultures grown for 4 or 8 days and in the presence or absence of the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3-ol (THIP). THIP (150 microM) induced a 2-fold increase in the number of alpha 1 and beta 2/3 subunits in both cell bodies and processes in 4-day-old cultures. Extending the culture period to 8 days led to a polarization of the receptor expression, since the increase in the number of subunits selectively was observed in the processes. Moreover, a general subcellular differentiation of the receptor population was observed in all culture conditions, since the ratio between the two subunits (beta 2/3; alpha 1) was four times higher in cell bodies compared to processes. A detailed analysis of the less mature (4-day-old) cultures revealed the existence of two populations of neurons exhibiting differences in the average number of receptors. During maturation neurons with few receptors developed into cells with a higher density of receptors resulting in a single population of the latter neurons, a process enhanced by exposure to THIP. This may indicate that receptor development is a discontinuous process with individual neurons following different temporal patterns. In double-labeling experiments, a spatially close association of the alpha 1 and beta 2/3 subunits could be seen, but the subunits were more frequently found separated from each other. In spite of the fact that exposure of the neurons to THIP increased the total number of receptor subunits, its presence apparently prevented formation of receptors with this subunit composition. Interestingly, receptor subunit clusters, consisting of alpha 1 alone, were more frequently observed than composite (alpha 1; beta 2/3) clusters. This substantiates the view that receptors not having alpha 1 and beta 2/3 subunits in the same complex may exist.

Animals↗

PubMed1994

1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice.

Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due to a transcriptional down-regulation.

Age Factors↗

PubMed1994

Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are enterocyte-specific gene products. The identification of regulatory cis-elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal-specific nuclear factors (NF-LPH1 and SIF1-BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis-elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 kDa oligomeric complexes. Based on these results NF-LPH1 and SIF1-BP are suggested to be either identical or closely related molecules.

Base Sequence↗

PubMed1994

On the transfer of serum proteins to the rat intestinal juice.

The in vivo pattern of serum proteins in the rat small-intestinal juice was characterized by crossed immunoelectrophoresis. Immunoglobulins and albumin, alpha-1-antitrypsin, transferrin, and orosomucoid were present. Larger serum proteins were absent (ceruloplasmin, haptoglobin, alpha-1-macroglobulin, alpha and beta lipoproteins). Thus, apart from immunoglobulins, only serum proteins with a molecular mass less than approximately 100 kDa were demonstrated. The origin and epithelial transfer were further characterized, using albumin as a model. No sign of local synthesis of albumin by the enterocytes was found by Northern blotting, and no albumin was found in the Golgi complex by immunogold electron microscopy. By immunogold electron microscopy a heavy labelling of albumin was observed in the interstitial spaces between the villus enterocytes. Where the enterocytes disintegrated, albumin was seen to leak out into the intestinal lumen from the opened interstitial spaces. A weak labelling was also found in the lysosomal/endosomal-like structures, especially in the crypt enterocytes, indicating pinocytosis of albumin. We conclude that the main reason for the occurrence of certain serum proteins in the intestinal juice is a selective passage through the capillary wall followed by passive intercellular transport via delivery of the serum in the interstitial space during disintegration of the enterocytes.

Animals↗

PubMed1994

Distribution of three microvillar enzymes along the small intestinal crypt-villus axis.

Aminopeptidase N (ApN), dipeptidyl peptidase IV (DPP IV) and sucrose-isomaltase (S-I) are differentially expressed along the pig jejunum crypt-villus axis. Quantitative immunoelectron microscopy and enzyme cytochemistry show that DPP IV and S-I are expressed in enterocytes along the entire length of the axis, whereas ApN is found mainly in the villar and upper crypt enterocytes. All three enzymes are detected in the basolateral membrane at all levels along the crypt-villus axis, although ApN and S-I only occurred at low intensities in the villus region. The microvillar/basolateral labelling ratio for the three enzymes increases to a varying degree for the three enzymes along the axis suggesting that the sorting efficiency to the apical membrane improves at least for ApN and S-I as the cells mature. These findings might indicate that the enterocytes change from a transcytotic to a direct apical transport as the enterocytes mature.

Animals↗

PubMed1993

Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells.

Effects of polyamine depletion by alpha-difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles relevant for protein synthesis was observed upon culturing in the presence of 4,5,6,7-tetrahydro-isoxazole[5,4-c]pyridin-3-ol (THIP) for 4 days. The intracellular concentrations of putrescine, spermidine, and spermine in these cultures were similar to the concentrations of the polyamines observed in cultures grown in a plain culture medium for 1, 2, 3 or 4 days, respectively. Upon culturing in the simultaneous presence of THIP and DFMO, the concentrations of putrescine and spermadine were reduced to less than 20% of the levels in the controls. This depletion was associated with a severely impaired morphological development of the granule cell cultures. Thus, the number of neurite extending cells was reduced to 50% of the number in the control cultures upon culturing in the presence of DFMO alone or in combination with THIP. Moreover, the THIP mediated increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO.

Animals↗

PubMed1993

Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA.

Neurotransmitter release and changes in the concentration of intracellular free calcium ([Ca++]i) were studied in cultured GABAergic cerebral cortical neurons, from mice, upon depolarization with either an unphysiologically high potassium concentration (55 mM) or the physiological excitatory neurotransmitter glutamate (100 microM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [3H]GABA as well as in [Ca++]i. However, the basic properties of transmitter release and the increase in [Ca++]i under a variety of conditions were different during stimulation with K+ or glutamate. Potassium-evoked release of [3H]GABA consisted of two phases, a rapid, large and transient phase followed by a smaller, more persistent second phase. The rapid phase was inhibited (60%) by nocodazole which reduced the number of vesicles in the neurites by 80%. This rapid phase of the GABA release was also reduced by organic (verapamil) and inorganic (Co++) Ca++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca++ channel antagonists but could be inhibited by SKF 89976A. The glutamate-induced [3H]GABA release, which was mainly mediated by N-methyl-D-aspartate receptors, consisted of a single, sustained phase. This was insensitive to nocodazole, partly inhibited by verapamil and could be blocked by Co++ as well as SKF 89976A. The action of Co++ could be attributed to a block of N-methyl-D-aspartate-associated ion channels. These findings strongly suggest that the majority of the K(+)-stimulated GABA release is dependent upon vesicles whereas the glutamate induced release is non-vesicular and mediated by a depolarization-dependent reversal of the direction of high-affinity GABA transport. The basic differences in the mode of action of the two depolarizing stimuli were reflected in the properties of the increase in [Ca++]i elicited by 55 mM K+ and 100 microM glutamate, respectively. The K(+)-induced increase in [Ca++]i was reduced by both verapamil and Ca(++)-free media whereas the corresponding glutamate response was only sensitive to Ca(++)-free conditions. Exposure of the cells to nocodazole or SKF 89976A had no effect on the ability of K+ or glutamate to increase [Ca++]i. Altogether, the results clearly demonstrate that K(+)-induced transmitter release from these GABAergic neurons is vesicular in nature whereas that induced by the neurotransmitter glutamate is not.

2-Amino-5-phosphonovalerate↗