5 RESULTS
5.1 Functional expression of the oxytocin system in primary human dermal
5.1.1 Expression of oxytocin and the oxytocin receptor
To determine if the OXT system is expressed by primary human dermal fibroblasts and keratinocytes, skin cells and suction-blister-roof-derived epidermal tissue were subjected to RTq-PCR analyses.
Analyses revealed that both, OXT and the OXTR, are expressed in dermal fibroblasts, keratinocytes and epidermis (Figure 12a, b). The highest mRNA expression levels of OXT and its receptor were detected in fibroblasts compared to keratinocytes or epidermal tissue.
Apart from this, OXT and OXTR expression could be also detected in other primary skin cells and cell lines like primary human melanocytes, human dermal microvascular endothelial cells (HDMEC) and the human mast cell line-1 (Figure 12c, d). Notably, porcine dorsal root ganglia neurons with attached Schwann cells also expressed OXT and its receptor. HDMECs showed the highest expression levels of OXT and the second highest expression of the
OXTR compared to all other tested cells (Figure 12a-d). The paraventricular nucleus and the supraoptic nucleus of the hypothalamus are the main expression sites of the OXT system.
Therefore, hypothalamus mRNA served as positive control for OXT/OXTR expression.
Figure 12. Expression of the oxytocin system in human skin cells.
(a, b) Total RNA isolated from dermal fibroblasts (F) (n = 81), keratinocytes (K) (n = 28) and suction-blister-roof derived epidermis (E) (n = 12) was subjected to reverse transcription. Resulting cDNA was analyzed for (a) OXT and (b) OXTR expression by RTq-PCR. (c, d) Total RNA isolated from melanocytes (M) (n = 6), human dermal microvascular endothelial cells (HDMEC) (n = 6), human mast cell line-1 (HMC-1) (n = 1), porcine dorsal root ganglia neurons with attached Schwann cells (N + S) (n = 1) and hypothalamus (HYPO) (n = 1) was subjected to reverse transcription. Resulting cDNA was analyzed for (c) OXT and (d) OXTR expression by RTq-PCR. Mann-Whitney U test, **P < 0.01,
***P < 0.001.
Immunofluorescence analyses confirmed OXT and OXTR expression in dermal fibroblasts and keratinocytes (Figure 13a-d). The staining displayed a diffuse distribution of OXT in both cell types (Figure 13a, c). Although the OXTR was also homogeneously spread in fibroblasts (Figure 13b), its presence in keratinocytes rather followed fibrous structures (Figure 13d).
Results 54 Both, OXT and the OXTR, seem to accumulate around the nuclei in dermal fibroblasts and keratinocytes (Figure 13a-d). Nevertheless, this observation needs to be confirmed by analyses using a confocal microscope.
In cryosections of skin biopsies, OXT was localized throughout the epidermis (Figure 13e). In contrast, the OXTR was mainly detected in the basal layer of the epidermis (Figure 13f).
Specificity of the secondary antibodies could be varified because no staining was detected in the samples in which the primary antibodies have been omitted (control) (Figure 13).
Figure 13. Immunofluorescent localization of the oxytocin system in human skin cells.
(a-f) Immunofluorescent detection of OXT (green) and the OXTR (green) was performed on dermal fibroblasts (F), keratinocytes (K) and the epidermis (E) of a skin cryosection. Immunofluorescence labeling procedure omitting the primary antibody served as negative control (Control) for specificity of the secondary antibody. Blue staining with DAPI depicts nuclei. Scale bar = 20 µM.
As the OXTR-directed antibody SYC592 was customized at the end of this thesis, its specificity has not been shown in publications. To demonstrate that the antibody SYC592 does specifically bind to the OXTR, a control experiment was performed using a blocking peptide. The blocking peptide served as antigen in the course of the production of SYC592 and corresponds to the OXTR epitope recognized by this antibody. Prior to the staining procedure, the antibody SYC592 was incubated with a 20-fold epitope masking excess of the blocking peptide (control).
Immunofluorescence analyses revealed that this pre-incubation step led to the neutralization of the antibody SYC592, showing its strong binding capacity to the OXTR (Figure 14a). To confirm this result, Western blot (WB) analysis using the SYC592 antibody for OXTR detection in fibroblasts 144 h post transfection with either scrambled or OXTR-specific siRNA was performed. After OXTR knockdown, the protein band corresponding to the OXTR was diminished (Figure 14b). A densitometric analysis of the OXTR amounts, using actin for normalization, revealed a reduced OXTR expression by 30 % and 60 % (Figure 14b). Thus, WB and its densitometric analysis confirmed the specificity of the antibody SYC592, albeit this result needs to be reproduced as only preliminary data are shown (n = 1, two experiments).
Figure 14. Testing of the antibody SYC592 for its OXTR-antigen specificity.
(a) Immunofluorescent detection of the OXTR (green) was performed on the epidermis of a skin cryosection. To demonstrate that the antibody SYC592 does specifically bind to the OXTR, a peptide blocking experiment was performed. Therefore, the antibody was pre-incubated with an excess of a peptide corresponding to the OXTR epitope recognized by the antibody. The resulting neutralized antibody (Control) was then used for immunofluorescence analysis. Blue staining with DAPI depicts nuclei. Scale bar = 20 µM. (b) WB analysis using the antibody SYC592 for OXTR detection in fibroblasts (n = 1, two experiments) 144 h post transfection with either scrambled (siControl) or OXTR-specific siRNA (siOXTR). Prior to WB, membrane fractions of lysates were subjected to SDS-PAGE (12 µg protein/lane). Actin served as loading control and for normalization in the densitometric analysis.
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5.1.2 Determination of non-toxic doses for the experimental use of
oxytocin and the oxytocin receptor antagonist L371,257
The neutral red assay was performed to determine non-toxic doses for OXT and for the OXTR antagonist L371,257 which were used in the consecutive in vitro experiments.
Doses of OXT between 1 nM and 1 µM did not influence cell viability of fibroblasts or keratinocytes (Figure 15a, b). Additionally, treatment of keratinocytes with 10 µM OXT did not affect cell viability neither (Figure 15b).
Figure 15. Cell viability of oxytocin-treated dermal fibroblasts and keratinocytes.
(a) Dermal fibroblasts (n = 6) and (b) keratinocytes (n = 6) were treated with increasing OXT concentrations for 24 h and subjected to the neutral red uptake assay to estimate the number of viable cells in culture. This assay is based on the ability of viable cells to incorporate and bind the dye neutral red in the lysosomes.
Doses of the OXTR antagonist L371,257 between 10 nM and 1 µM did not compromise cell viability of fibroblasts or keratinocytes (Figure 16a, b). Whereas also no cytotoxic effects on keratinocytes were measured after treatment with 10 µM antagonist, a marginal decrease by 7 ± 2 % in cell viability was detected in fibroblasts (Figure 16a). This was due to the antagonist resolvent DMSO which slightly reduced (by 11 ± 1 %) cell viability as well. Based on these results, concencentrations of OXT from 1 nM to 1 µM for the treatment of fibroblasts and from 1 nM to 10 µM for the treatment of keratinocytes were used in the in vitro experiments. Applicable doses of the OXTR antagonist range from 10 nM to 1 µM for the treatment of fibroblasts and from 10 nM to 10 µM for the treatment of keratinocytes.
Figure 16. Cell viability of dermal fibroblasts and keratinocytes after treatment with the oxytocin receptor antagonist L371,257.
(a) Dermal fibroblasts (n = 6) and (b) keratinocytes (n = 6) were treated with increasing concentrations of the OXTR antagonist L371,257 for 24 h and subjected to the neutral red uptake assay to estimate the number of viable cells in culture. As the OXTR antagonist is dissolved in DMSO, equal volumes of DMSO for the indicated antagonist concentrations were used. Wilcoxon signed-rank test, Paired t-test,
*P < 0.05.
5.1.3 siRNA-mediated knockdown of the oxytocin receptor
To investigate cellular functions of the OXT system in dermal fibroblasts and keratinocytes, siRNA-mediated knockdown of the OXTR was established.
First, three siRNAs (I = ID1859, II = ID1766, III = ID143368, Applied Biosystems) with different sequences directed against the OXTR (siOXTR) were tested for knockdown efficiency in dermal fibroblasts. RTq-PCR analysis revealed that all tested siRNAs were suitable to knockdown the OXTR (Figure 17a). Expression of the OXTR was reduced to 0.24 with siOXTR I, to 0.07 with siOXTR II and to 0.11 with siOXTR III relative to untreated control. siOXTR II was the most potent siRNA to achieve OXTR knockdown and therefore used in all consecutive experiments. Neither the transfection reagent (Lipofectamine® RNAiMAX) nor the scrambled siRNA (1027281, Qiagen) did remarkably influence OXTR expression (0.92 and 0.97, respectively) relative to untreated control. Thus, both were considered to be appropriate for further use. Next, the persistence of knockdown was determined. Knockdown of the OXTR was stable for all tested time points ranging from 24 to 144 h post transfection (Figure 17b).
Results 58
Figure 17. Efficiency of siRNA for oxytocin receptor knockdown and kinetics of knockdown in dermal fibroblasts.
(a) To identifiy the most effective siRNA for OXTR knockdown, dermal fibroblasts (n = 1) were transfected with either 50 nM of scrambled siRNA, 50 nM of three different OXTR-specific siRNAs (siOXTR I, II and III), treated with transfection reagent alone or left untreated. Cells were harvested 48 h post transfection and OXTR expression relative to untreated control was analyzed by RTq-PCR.
(b) To determine the duration of OXTR knockdown in dermal fibroblasts (n = 2-4), cells were harvested at different time points post transfection with 50 nM of siOXTR II or scrambled siRNA. They were subjected to RTq-PCR and OXTR expression relative to untreated control was analyzed.
In order to minimize non-specific effects due to high intracellular siRNA levels, it is beneficial to reduce siRNA concentrations if possible. For this purpose, it was tested wether the previously used siRNA concentration of 50 nM could be reduced to 20 nM. RTq-PCR analysis revealed no remarkable differences in OXTR knockdown efficiency in fibroblasts after transfection with 20 nM and with 50 nM siRNA (Figure 18a). Thus, the lower siRNA concentration was consecutively used for OXTR knockdown in fibroblasts.
An analogues approach to that for fibroblasts was performed to optimize the transfection conditions for keratinocytes (data not shown). The final protocol used for OXTR knockdown in fibroblasts and keratinocytes is described in section 4.3.3. Ultimately, the optimized transfection conditions for effective OXTR knockdown in fibroblasts and keratinocytes were approved by RTq-PCR (Figure 18b). 120 h post transfection, knockdown reduced OXTR expression in dermal fibroblasts to 0,07 ± 0,02 and in keratinocytes to 0,28 ± 0,03 relative to scrambled control.
Figure 18. Testing of siRNA concentrations and the optimized conditions for oxytocin receptor knockdown in dermal fibroblasts and keratinocytes.
(a) To determine suitable siRNA concentrations for OXTR knockdown, dermal fibroblasts (n = 3-8) and were transfected with either scrambled siRNA or OXTR-specific siRNA (siOXTR II). 20 nM and 50 nM of siRNA were used for transfection. Cells were harvested 72 h post transfection and OXTR expression relative to scrambled control (red line) was analyzed by RT-PCR. (b) OXTR expression relative to scrambled control (red line) in dermal fibroblasts (n = 8) and keratinocytes (n = 4) 120 h post transfection with either scrambled siRNA or OXTR-specific siRNA was assessed by RT-PCR.
Paired t-test, ***P < 0.001.
5.1.4 Functionality of the oxytocin receptor measured by intracellular calcium fluxes
The OXTR predominantly couples to Gq-proteins which trigger inositol-3-phosphate signalling, leading to increased intracellular Ca2+ fluxes (2). In order to test, whether the OXTR is functional in dermal fibroblasts and keratinocytes, Ca2+fluxes were measured after activation with OXT. For this purpose, cells were stained with two fluorescent dyes, Fluo-4 and Fura-Red. Upon Ca2+binding, excitement at 488 nm leads to an increase in green fluorescence (525 nm) emitted by Fluo-4, whereas red fluorescence (640 nm) emitted by Fura-Red decreases. Based on this, Ca2+ fluxes which are proportional to the Fluo-4/Fura-Red ratio were monitored by flow cytometry.
In both cell types, stimulation with OXT led to a significant increase in Ca2+currents in a dose-dependent manner, indicating that the OXTR is functional (Figure 19a, d). Notably, fibroblasts were less sensitive to low and middle OXT concentrations compared to keratinocytes. Stimulation with 1 nM OXT was not sufficient to induce Ca2+ fluxes (1.1 ± 0.1) in fibroblasts, whereas the same OXT concentration significantly induced Ca2+ fluxes (1.7 ± 0.1) in keratinocytes. Although stimulation with 10 nM and 100 nM OXT also significantly increased Ca2+ fluxes (1.3 ± 0.1 and 1.8 ± 0.2) in fibroblasts, Ca2+ fluxes induced by these OXT concentrations were still higher (2 ± 0.3 and 2.3 ± 0.3) in keratinocytes. The
Results 60 amplitude of Ca2+ currents induced by a high OXT concentration (1 µM) was equal in both cell types (fibroblasts: 2.1 ± 0.3 and keratinocytes: 2.2 ± 0.2). In keratinocytes, saturation seems to occure at an OXT concentration of 100 nM. Because no difference in the amplitude of the Ca2+ response induced by 100 nM OXT and 1 µM OXT was detected.
Specificity of the observed Ca2+ responses upon OXT stimulation was confirmed by OXTR knockdown in fibroblasts and the use of the OXTR antagonist L371,257 in keratinocytes. No Ca2+ fluxes in response to OXT stimulation were detected in OXTR knockdown fibroblasts.
Compared to OXTR knockdown, the use of the OXTR antagonist was less effective to inhibit OXT-induced Ca2+ fluxes. Nevertheless, for keratinocytes, its use as a negative control was more appropriate than OXTR knockdown, because all transfected keratinocytes exhibited damped Ca2+ fluxes (data not shown). Overall, both negative controls significantly inhibited OXT-induced Ca2+ fluxes.
Visualization of OXT-induced Ca2+ fluxes via fluorescence analyses confirmed OXTR signalling in dermal fibroblasts and keratinocytes (Figure 19b, e). The change of color after OXT stimulation from red/orange to green/yellow indicates occurence of intracellular Ca2+
fluxes.
Ionomycin was used as a positive control to obtain a maximum calcium influx. Ionomycin-stimulated fibroblasts exhibited a fivefold induction of Ca2+ influx, whereas a twofold induction was detected in keratinocytes (Figure 19c). Both, stimulation with OXT and ionomycin, led within five seconds to a maximal increase in Ca2+ influx in dermal fibroblasts (Figure 19f).
The kinetic course was similar, although the amplitude of Ca2+ influx was extenuated after OXT stimulation.
Figure 19. Oxytocin-induced Ca2+ signalling in dermal fibroblasts and keratinocytes.
(a, d) OXT-induced intracellular Ca2+ fluxes relative to baseline (set at 1) were measured in (a) dermal fibroblasts (n = 6) and (b) keratinocytes (n = 6). siRNA-mediated knockdown of the OXTR (siOXTR) in fibroblasts and the treatment of keratinocytes with the OXTR antagonist L371,257 served as negative controls. (b, e) Representative images of OXT induced Ca2+ fluxes (green/yellow). (c) Ionomycin-induced intracellular Ca2+ fluxes relative to baseline (set at 1) were measured in dermal fibroblasts (F) (n = 6) and keratinocytes (K) (n = 6). (f) Representative kinetics of Ionomycin- and OXT-induced Ca2+
fluxes in dermal fibroblasts. Paired t-test, *P < 0,05, **P < 0,01, ***P < 0,001.
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5.2 Modulation of the oxytocin system in human skin
5.2.1
Modulation of oxytocin and oxytocin receptor expression in dermal fibroblasts and keratinocytes
in vitroIn order to investigate whether OXTR mRNA levels are altered in dermal fibroblasts and keratinocytes upon exposure to OXT, RTq-PCR analyses were performed. The standard culture medium for fibroblasts contains FCS-derived OXT at a concentration of 66 pM, whereas the standard medium for keratinocytes contains bovine pituitary extract (BPE)-derived OXT at a concentration of 2.7 nM. To examine, if cell culture OXT concentrations affect responsiveness to OXT-treatment, standard culture conditions were compared with serum-reduced culture conditions. For serum-reduced conditions, culture media without BPE or with a lower FCS content (0,2 %) was used.
OXTR expression was significantly increased (2.7 ± 0.3) in fibroblasts, kept in standard culture medium containing 10 % FCS, after 12 h of continuous OXT-treatment (Figure 20a).
In contrast, when the culture medium contained only 0.2 % FCS, the OXT-induced increase of OXTR expression (4.3 ± 2.6) was detected after 18 h (Figure 20b). Differences in the amplitudes at both maxima of OXTR expression were not significant. In addition, OXT treatment did not influence OXTR expression in fibroblasts at any other tested time point, before or after 12 h and 18 h, respectively.
In keratinocytes, OXT treatment did not induce significant changes in OXTR expression levels (Figure 20c, d). Neither keratinocytes kept in standard culture medium containing BPE, nor keratinocytes kept in culture medium without BPE, showed remarkable alterations of OXTR expression at any tested time point.
Figure 20. Oxytocin receptor expression in oxytocin-treated dermal fibroblasts and keratinocytes under different serum culture conditions.
(a-d) Transcript levels of the OXTR in dermal fibroblasts and keratinocytes were assessed by RT-PCR. (a, b) Fibroblasts (n = 3), kept in culture medium containing (a) 10 % FCS or (b) 0,2 % FCS, were left untreated or treated with 100 nM OXT for the time period indicated and harvested directly after treatment. (c, d) Keratinocytes (n = 3), kept in culture medium containing (c) bovine pituirary extract (BPE) or (d) without BPE, were left untreated or treated with 100 nM OXT for the time period indicated and harvested directly after treatment. Paired t-test, *P < 0,05.
Next, OXT expression levels were investigated in OXTR knockdown dermal fibroblasts and keratinocytes 120 h post transfection. OXT expression in OXTR-depleted fibroblasts was significantly reduced (0.6 ± 0.03) relative to control cells (Figure 21a). In contrast, OXTR knockdown keratinocytes did not exhibit any change in OXT expression compared to control (Figure 21b).
Results 64
Figure 21. Oxytocin expression in oxytocin receptor-depleted dermal fibroblasts and keratinocytes.
(a, b) Expression of OXT in OXTR knockdown (a) fibroblasts (n = 4) and (b) keratinocytes (n = 4) relative to control. Cells were kept in standard culture medium, harvested 120 h post transfection and OXT transcript levels were assessed by RT-PCR. Paired t-test, **P < 0,01.
Furthermore, organotypic 3-dimensional (D) skin cultures were constructed to examine whether OXT treatment influences OXT expression in an epidermal tissue. The advantage of 3-D skin cultures is that they constitute a more physiological environment than 2-D monocultures. In these skin models, the keratinocytes develop an in vivo-like epidermal stratified architecture upon air exposure (78). The organotypic cultures were daily treated with 1 µM OXT, harvested at day 15 and OXT expression was assessed by RTq-PCR.
Here, OXT treatment led to a significant increase (5.3 ± 1.3) of OXT expression relative to control (Figure 22).
Figure 22. Oxytocin expression in the epidermis of an oxytocin-treated organotypic 3-D skin model.
Organotypic cultures were daily treated with OXT. At day 15, the epidermis (n = 4) of these cultures was harvested. Subsequently, transcript levels of OXT relative to control were assessed by RT-PCR.
Paired t-test, *P < 0,05.
5.2.2
Modulation of oxytocin release in the skin
in vivoStudies have shown that plasma and salivary levels of OXT increase in response to warm partner touch (83, 84). To examine whether OXT might be locally released from human skin in vivo, a study with 12 healthy volunteers, including six women and six men, was performed.
OXT concentrations in suction blister fluids (SBFs), taken from both arms of the volunteers were measured by ELISA technique. At t0, no treatment was performed. At t1, participants caressed their arm with a soft brush around the investigated area (tactile stimulation) leaving the other arm unstimulated (control).
Analyses of SBFs taken at t0 demonstrated that with respect to gender, OXT levels were equivalent (males: 6.5 ± 0.6 pg OXT/mg protein, females: 6.4 ± 1 pg OXT/mg protein) (Figure 23a). Regarding OXT concentrations in SBFs taken at t1 revealed that tactile stimulation of the skin triggers local OXT release. Significantly higher OXT concentrations were observed in SBFs taken from stimulated arms (6.8 ± 0.7 pg OXT/mg protein) compared to unstimulated arms (5.9 ± 0.4 pg OXT/mg protein) (Figure 23b). Overall, the OXT contents in SBF samples ranged from 70 to 160 pM.
Results 66
Figure 23. Oxytocin concentrations in suction blister fluids after tactile stimulation.
(a, b) OXT concentrations in human skin suction blister fluids (SBF) taken from both arms of male (n = 6) and female (n = 6) volunteers were measured by ELISA-technique. (b) OXT concentrations in SBF derived from tactile-stimulated and control areas. Wilcoxon signed-rank test, *P < 0.05.
5.3 Comparison of the expression of the oxytocin system in healthy and atopic skin cells
To explore whether the OXT system might be dysregulated in atopic skin, the expression of OXT and the OXTR in dermal fibroblasts and keratinocytes, derived from biopsies of healthy volunteers and atopic skin patients, were examined.
RTq-PCR analyses revealed significantly reduced OXT expression in atopic lesional fibroblasts by 2.8 ± 2.6 and in atopic peri-lesional keratinocytes by 0.9 ± 0.1 compared to healthy controls (Figure 24a). Additionally, OXT concentrations in culture supernatants of atopic lesional fibroblasts were reduced by 5.9 ± 2 pg/ml compared to healthy control cells (Figure 24c). Atopic peri-lesional fibroblasts and atopic lesional keratinocytes also showed reduced OXT expression. Moreover, OXTR expression was significantly decreased in both, atopic peri-lesional and lesional fibroblasts (by 620 ± 52, 560 ± 65, respectively), compared to healthy fibroblasts (Figure 24b). Atopic keratinocytes also showed a decreased OXTR expression (Figure 24b). Accordingly, a similar trend to reduced OXTR expression levels was detected in epidermis derived from atopic peri-lesional suction blister roofs (Figure 24b). To evaluate OXT levels in atopic versus healthy skin in vivo, OXT concentrations in suction blister fluids were measured. Suction blister fluids derived from peri-lesional atopic skin exhibited a slight decrease of OXT concentration compared to the healthy control (Figure 24d). These data suggest that atopic skin displays a deficit in OXT signalling.
Figure 24. Expression of oxytocin and the oxytocin receptor in atopic skin cells.
(a, b) Transcript levels for OXT and the OXTR in healthy (h), peri-lesional (pl) and lesional (l) atopic (a) dermal fibroblasts (F), keratinocytes (K) and suction-blister-roof-derived epidermis (E) were assessed by RT-PCR. (c) OXT concentrations in culture supernatants of lesional atopic and healthy fibroblasts 48 h after addition of fresh media. (d) OXT concentrations in suction blister fluids (SBF) of peri-lesional atopic and healthy skin were detected by ELISA-technique. Unpaired t-test, Mann–Whitney U test,
*P < 0.05, **P < 0.01.
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5.4 Cellular functions of the oxytocin system in primary human dermal fibroblasts and keratinocytes
To elucidate OXTR-mediated functions in the skin, transient siRNA-mediated OXTR knockdown experiments were performed. Subsequently, neuroendocrine, inflammatory and oxidative stress parameters were measured and OXT-induced effects on cell growth and epidermal structure were investigated.
5.4.1 Effects of oxytocin receptor knockdown on the neuroendocrine stress mediators CRH and corticosterone
Corticotropin-releasing hormone (CRH) is one of the key players mediating neuroendocrine stress responses. Moreover, its receptor (CRHR) is inhibited by OXT in term myometrium (85). To examine whether the OXT system might modulate CRH and CRHR1 in skin cells, their expression in OXTR knockdown dermal fibroblasts and keratinocytes was analyzed via RTq-PCR.In fibroblasts, knockdown of the OXTR led to a fivefold upregulation of CRH and a 39-fold upregulation of CRHR1 expression 96 h post transfection (Figure 25a, b). In keratinocytes, no CRH and CRHR1 expression was detected, neither in control cells nor after OXTR knockdown (data not shown).
Figure 25. Expression of corticotropin-releasing hormone and its receptor after oxytocin receptor knockdown in dermal fibroblasts.
(a, b) Transcript levels of CRH (a) and CRHR1 (b) in dermal fibroblasts (n = 3) transfected with either scrambled siRNA (control) or OXTR-specific siRNA (siOXTR). Cells were harvested 96 h post transfection and gene expressions were assessed by RT-PCR.
Corticosterone is released in response to stress and its production in the skin can be regulated by CRH (86). To evaluate whether the OXT system might affect corticosterone release from skin cells, corticosterone levels in the supernatants of OXT-treated dermal fibroblasts and keratinocytes were measured by ELISA technique.
Corticosterone concentrations in the supernatants of fibroblasts were below detection thresholds (data not shown). Supernatants of OXT-treated keratinocytes contained
Corticosterone concentrations in the supernatants of fibroblasts were below detection thresholds (data not shown). Supernatants of OXT-treated keratinocytes contained