Transferrin receptor expression and role in transendothelial transport of transferrin in cultured... more Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers, Molecular and Cellular Neuroscience (2016), Abstract A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 2
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calc... more The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
Journal of the Marine Biological Association of the United Kingdom, 1997
ABSTRACT Rhizocephalans are highly specialized crustaceans adapted for parasitic life on decapods... more ABSTRACT Rhizocephalans are highly specialized crustaceans adapted for parasitic life on decapods. The juvenile parasite passes through an internal growth stage, where a root-like trophic organ invades the tissue and haemocoelic fluid of the host (for detailed information on rhizocephalan life history, see Haeg, 1992). As in free-living barnacles, a cyprid larva is the settling stage, and in thoracicans a juvenile barnacle is formed underneath the carapace of the cyprid larva. However, the rhizocephalans develop a so-called kentrogon, which accomplishes the injection of the primordial parasite (Glenner & Høeg, 1995). The degree of specialization towards a specific host means that the cypris larvae of rhizocephalans are extremely selective in their choice of substrata. They will not settle unless a specific host or part of a host, e.g. the gills, is offered. For this reason, the study of settlement and metamorphosis of these species has met with great difficulties.
Pfl�gers Archiv European Journal of Physiology, 1993
The aim of the present study was to investigate the effects of small, unilateral changes in solut... more The aim of the present study was to investigate the effects of small, unilateral changes in solution osmolarity on active sodium transport and cellular electrophysiological parameters in frog skin. The active sodium transport across the skin was measured as the amiloride-sensitive short-circuit current (Isc) and cellular potential was monitored with microelectrodes, while small (+_ 20 mOsm) osmotic gradients were imposed on the skin. Increasing the osmolarity of the apical bathing solution (or decresing the osmolarity of the basolateral solution) increased/so lowered tissue resistance (R), depolarized the cellular potential and decreased the fractional resistance of the apical membrane, which indicates an increased apical sodium permeability. Conversely, a similar increase in basolateral osmolarity (or a decrease in apical osmolarity) lowered the Lc, increased R, hyperpolarized the cells and increased the fractional resistance of the apical membrane, indicating a decrease in apical sodium permeability. The results indicate that the osmotic gradient across the skin, rather than solution osmolarity as such, is responsible for the observed changes in/~o and apical sodium permeability after small osmotic perturbations.
Pfl�gers Archiv European Journal of Physiology, 1999
In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in t... more In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na+ transport. Cytosolic calcium ([Ca2+]i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na+ transport was measured as the amiloride-sensitive short-circuit current (Isc) using a conventional voltage clamp. Basal [Ca2+]i was 65+/-6 nM (n=15). Removal of Na+ from the mucosal solution had no effect on [Ca2+]i. When Na+ was removed from the serosal solution, [Ca2+]i increased biphasically to a peak of 220+/-38 nM (n=8, P=0.006). Readdition of Na+ to the serosal solution returned [Ca2+]i to control level. The serosal Na+ gradient and changes in [Ca2+]i were closely correlated; stepwise changes in serosal Na+ were followed by stepwise changes in [Ca2+]i. These observations indicate the existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na+ transport decreased from 13.2+/-1.8 to 9.2+/-1.5 microA cm-2 (n=8, P=0.049) when Na+ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca2+, Na+ transport decreased similarly from 16.7+/-1.7 to 11.6 +/-1. 8 microA cm-2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in Isc after removal of serosal Na+ is due to an increase in [Ca2+]i per se.
Pfl�gers Archiv European Journal of Physiology, 1996
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calc... more The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
Pfl�gers Archiv European Journal of Physiology, 1994
Cytosolic calcium ([Ca2+]i) has been suggested as a key modulator in the regulation of active sod... more Cytosolic calcium ([Ca2+]i) has been suggested as a key modulator in the regulation of active sodium transport across electrically "tight" (high resistance) epithelia. In this study we investigated the effects of calcium on cellular electrophysiological parameters in a classical model tissue, the frog skin. [Ca2+]i was measured with fura-2 in an epifluorescence microscope setup. An inhibition of basolateral potassium permeability was observed when cytosolic calcium was increased. This inhibition was reversible upon removal of calcium from the serosal solution.
Pfl�gers Archiv European Journal of Physiology, 2000
The present study presents the first evidence for P2Y-type adenosine 5'-triphosph... more The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.
Pfl�gers Archiv European Journal of Physiology, 2000
In the present study we investigated the effects of adenosine 5'-triphosphate (AT... more In the present study we investigated the effects of adenosine 5'-triphosphate (ATP) on Na+ transport in frog skin epithelium. An experimental set-up was constructed to allow simultaneous measurement of Na+ transport, measured as the amiloride-sensitive short circuit current (Isc), and free cytosolic Ca2+ concentration ([Ca2+]i) measured with the Ca(2+)-sensitive dye fura-2. The cell potential (Vsc) was measured with microelectrodes. Addition of ATP (100 micrM) to the basolateral solution resulted in a fast transient decrease in Isc followed by a slower increase and a transient increase in [Ca2+]i. Microelectrode measurements showed that the primary response, i.e. the decline in Isc was accompanied by transient depolarisation, followed by a return to the control value. The decrease in current was Ca2+ independent; i.e. treatment with thapsigargin in Ca(2+)-free solutions abolished the Ca2+ transient but did not influence the current transient. The secondary response, i.e. the slow increase in current, was accompanied by slow depolarisation of the cell. Measurements of apical Na+ permeability showed that this was due to an opening or activation of apical Na+ channels. These data show that ATP causes a fast initial drop and a secondary, long-lasting increase in Na+ absorption. The ability of ATP to cause the initial decline in current is independent of Ca2+, i.e. it is not caused by secondary effects of the P2Y-type receptors present in the tissue. Measurements of intracellular potential indicate that the initial depolarisation is caused by opening of non-selective cation channels, suggesting that this decrease is due to a transient activation of P2X-type ATP receptors.
The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides i... more The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of peptide transport activity in physiological and/or pathophysiological situations, where EGF and EGF receptor levels change drastically, remains to be established.
This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOM... more This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of −33 to −26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.
Transferrin receptor expression and role in transendothelial transport of transferrin in cultured... more Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers, Molecular and Cellular Neuroscience (2016), Abstract A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 2
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calc... more The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
Journal of the Marine Biological Association of the United Kingdom, 1997
ABSTRACT Rhizocephalans are highly specialized crustaceans adapted for parasitic life on decapods... more ABSTRACT Rhizocephalans are highly specialized crustaceans adapted for parasitic life on decapods. The juvenile parasite passes through an internal growth stage, where a root-like trophic organ invades the tissue and haemocoelic fluid of the host (for detailed information on rhizocephalan life history, see Haeg, 1992). As in free-living barnacles, a cyprid larva is the settling stage, and in thoracicans a juvenile barnacle is formed underneath the carapace of the cyprid larva. However, the rhizocephalans develop a so-called kentrogon, which accomplishes the injection of the primordial parasite (Glenner & Høeg, 1995). The degree of specialization towards a specific host means that the cypris larvae of rhizocephalans are extremely selective in their choice of substrata. They will not settle unless a specific host or part of a host, e.g. the gills, is offered. For this reason, the study of settlement and metamorphosis of these species has met with great difficulties.
Pfl�gers Archiv European Journal of Physiology, 1993
The aim of the present study was to investigate the effects of small, unilateral changes in solut... more The aim of the present study was to investigate the effects of small, unilateral changes in solution osmolarity on active sodium transport and cellular electrophysiological parameters in frog skin. The active sodium transport across the skin was measured as the amiloride-sensitive short-circuit current (Isc) and cellular potential was monitored with microelectrodes, while small (+_ 20 mOsm) osmotic gradients were imposed on the skin. Increasing the osmolarity of the apical bathing solution (or decresing the osmolarity of the basolateral solution) increased/so lowered tissue resistance (R), depolarized the cellular potential and decreased the fractional resistance of the apical membrane, which indicates an increased apical sodium permeability. Conversely, a similar increase in basolateral osmolarity (or a decrease in apical osmolarity) lowered the Lc, increased R, hyperpolarized the cells and increased the fractional resistance of the apical membrane, indicating a decrease in apical sodium permeability. The results indicate that the osmotic gradient across the skin, rather than solution osmolarity as such, is responsible for the observed changes in/~o and apical sodium permeability after small osmotic perturbations.
Pfl�gers Archiv European Journal of Physiology, 1999
In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in t... more In the present study we investigated the possible existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na+ transport. Cytosolic calcium ([Ca2+]i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na+ transport was measured as the amiloride-sensitive short-circuit current (Isc) using a conventional voltage clamp. Basal [Ca2+]i was 65+/-6 nM (n=15). Removal of Na+ from the mucosal solution had no effect on [Ca2+]i. When Na+ was removed from the serosal solution, [Ca2+]i increased biphasically to a peak of 220+/-38 nM (n=8, P=0.006). Readdition of Na+ to the serosal solution returned [Ca2+]i to control level. The serosal Na+ gradient and changes in [Ca2+]i were closely correlated; stepwise changes in serosal Na+ were followed by stepwise changes in [Ca2+]i. These observations indicate the existence of a Na+/Ca2+ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na+ transport decreased from 13.2+/-1.8 to 9.2+/-1.5 microA cm-2 (n=8, P=0.049) when Na+ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca2+, Na+ transport decreased similarly from 16.7+/-1.7 to 11.6 +/-1. 8 microA cm-2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in Isc after removal of serosal Na+ is due to an increase in [Ca2+]i per se.
Pfl�gers Archiv European Journal of Physiology, 1996
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calc... more The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
Pfl�gers Archiv European Journal of Physiology, 1994
Cytosolic calcium ([Ca2+]i) has been suggested as a key modulator in the regulation of active sod... more Cytosolic calcium ([Ca2+]i) has been suggested as a key modulator in the regulation of active sodium transport across electrically "tight" (high resistance) epithelia. In this study we investigated the effects of calcium on cellular electrophysiological parameters in a classical model tissue, the frog skin. [Ca2+]i was measured with fura-2 in an epifluorescence microscope setup. An inhibition of basolateral potassium permeability was observed when cytosolic calcium was increased. This inhibition was reversible upon removal of calcium from the serosal solution.
Pfl�gers Archiv European Journal of Physiology, 2000
The present study presents the first evidence for P2Y-type adenosine 5'-triphosph... more The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.
Pfl�gers Archiv European Journal of Physiology, 2000
In the present study we investigated the effects of adenosine 5'-triphosphate (AT... more In the present study we investigated the effects of adenosine 5'-triphosphate (ATP) on Na+ transport in frog skin epithelium. An experimental set-up was constructed to allow simultaneous measurement of Na+ transport, measured as the amiloride-sensitive short circuit current (Isc), and free cytosolic Ca2+ concentration ([Ca2+]i) measured with the Ca(2+)-sensitive dye fura-2. The cell potential (Vsc) was measured with microelectrodes. Addition of ATP (100 micrM) to the basolateral solution resulted in a fast transient decrease in Isc followed by a slower increase and a transient increase in [Ca2+]i. Microelectrode measurements showed that the primary response, i.e. the decline in Isc was accompanied by transient depolarisation, followed by a return to the control value. The decrease in current was Ca2+ independent; i.e. treatment with thapsigargin in Ca(2+)-free solutions abolished the Ca2+ transient but did not influence the current transient. The secondary response, i.e. the slow increase in current, was accompanied by slow depolarisation of the cell. Measurements of apical Na+ permeability showed that this was due to an opening or activation of apical Na+ channels. These data show that ATP causes a fast initial drop and a secondary, long-lasting increase in Na+ absorption. The ability of ATP to cause the initial decline in current is independent of Ca2+, i.e. it is not caused by secondary effects of the P2Y-type receptors present in the tissue. Measurements of intracellular potential indicate that the initial depolarisation is caused by opening of non-selective cation channels, suggesting that this decrease is due to a transient activation of P2X-type ATP receptors.
The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides i... more The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of peptide transport activity in physiological and/or pathophysiological situations, where EGF and EGF receptor levels change drastically, remains to be established.
This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOM... more This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of −33 to −26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.
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