British journal of Dermatology {1992) 127. 595-599.
Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis H.KOIZUMI, H.TANAKA. T.FUKAYA AND A.OHKAWARA Department of Dermatology. Hokkaido University School of Medicine, Kita I 5 Nishi 7, Kita-Ku, Sapporo, 060, japan Accepted for publication JO April 1 992
Summary
Substance P is a neuropeptidc present in, and released from, peripheral C nerve endings. The presence of substance P-positive nerve fibres in the epidermis has been reported. We investigated the efTect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5-lrisphosphate (IPs), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the /i-adrenergic- or histamine (H2)- adenylate cyclase responses of the epidermis. Neither forskolin-induced. nor cholera toxininduced cyclic AMP accumulation were affected by substance P treatment. These results are consistent with the view that substance P stimulates phosphatidylinositol-4.S-bisphosphate (PlPi) hydrolysis of keratinocytes. resulting in IPj-Ca-* and diacylglycerol-protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyciase system, no evidence for such 'cross-talk regulation' was detected in keratinocytes by substance P treatment.
Substance P is a neuropeptide. which is present in unmyelinated nerve endings in the skin.'^ Substance Ppositive nerve endings have been demonstrated in the epidermis and dermis of normal and psoriatic skin, and psoriatic plaques are more densely innervated than normal skin.' The content of substance P is also reported to be elevated in psoriatic skin.'* Substance P is released from peripheral nerve endings by various stimuli. Farber et aV suggested that substance P might have pathophysiological significance in psoriasis. According to their view, substance P released from the peripheral nerve endings afTects various cell systems directly or indirectly, resulting in the development of psoriatic lesions. For example, substance P induces histamine release from mast cells, which results in cutaneous vasodilatation.'' and Tanaka et al7 reported that substance P increases Pam 212 keratinocyte proliferation. Although the latter finding has not been confirmed unequivocally by studies using normal human keratinocytes in culture."^ these findings could be related to the formation of the erythematous. hyperproliferative lesions in psoriasis. There is little additional information about other biological aspects of the effects of substance P on keratinocytes. Interestingly, substance P has been shown to stimulate phosphatidylinositol turnover and to Correspondence: Dr H.Koizumi.
activate protein kinase C in other cell systems.'" " The part played by these signalling systems in epidermal proliferation and differentiation, which are deranged in various pathological conditions ofthe epidermis, including psoriasis,''" is a matter of interest. Consequently, we investigated the effect of substance P on various transmembrane signalling systems in epidermal keratinocytes.
Methods Chemicals
Substance P. 12-o-tetradecanoylphorbol-1 3-acetate (TPA). phosphatidylserine. and cholera toxin were purchased from Sigma Chemical Co. (St Louis. MO. U.S.A.). (y-'^P)ATP and D-myo-inositol 1.4.5-trisphosphate (IPO [*H] assay system were purchased from Amersham |apan Corp. (Tokyo. Japan). Spantide was purchased from Bachem Inc. (CA. U.S.A.). Forskolin was obtained from Calbiochem Co. (La JoUa, CA. U.S.A.). Cyclic AMP assay kit is a product of Yamasa Shoyu Co. (Tokyo, Japan). Protein kinase C preparation
Skin slices were obtained from the backs of domestic pigs (weight 5-10 kg) with a Castroviejo keratome set at 0 2 595
596
H.KOIZUMI et al.
mm thickness. The skin slices were treated with 1000 U/ ml of dispase in RPMI 1640 medium for 30 min at 3 7°C. Following the dispase treatment, the pure epidermal sheets were peeled off with sharp forceps. The epidermal sheets were then floated on fresh RPMI 1640 medium for 30 min at 37°C. and were incubated with 50 fiM of substance P for 15 min. Following incubation, the epidermal sheets were wiped with filter paper, and immediately homogenized in a conical glass homogenizer with 5 vol (w/v) of 20 mM Tris HCl. pH 7-5, containing 0 25 M sucrose. 2 mM EDTA. 0-5 mM ECTA. and 2 mM phenylmethylsulphonyl fluoride (PMSF). The enzyme source was centrifuged at 105.000 g for 60 min. The supernatant and the pellet were used as the source of protein kinase C in the cytosol fraction and in the membrane fraction, respectively. These crude extracts were applied to a UE 52-cellulose column and the enzyme was eluted with 1 5 column volumes of 0-08 M NaCl. Protein kinase C activity was measured as previously described.'^
Detection of intracellular calcium alteration
Alteration of intracellular Ca-^^ of single living epidermal keratinocytes was measured by the method of Williams et al.^'' using a fluorescent Ca^+-sensitive dye. Fura 2AM and inverted fluorescence microscopy, as previously described.^^ Pure epidermal sheets were pre-incubated for 30 min. and then incubated with Fura 2-AM for 30 min. The keratinocytes were placed on the stage of an inverted fluorescence microscope. Fifty micromoles of substance P and 1 mM of histamine were added to the cells in succession. Five points of different cells were recorded simultaneously.
Measurement of inosito!
IPj content in the samples was measured by a radiochemical method using a D-myo-inositol 1.4.5-trisphosphate assay system. Ci/(7ic AMP accumulation
Pig epidermal slices obtained by dispase treatment were cut into pieces 5 mm^ with a razor blade, and were rinsed three times in RPMI 1640 medium. They were then floated on the buffer with the keratin layer uppermost. The epidermal slices were pre-incubated at 37°C for 20 min in RPMI 1640 medium in the water bath. For receptor-stimulated cyclic AMP accumulation. the specimens were incubated at 37°C for 5 min in the RPMI 1640 medium with 50/iM epinephrine or 0-1 mM histamine, in the presence or absence of 50 fiM of substance P. The slices were quickly frozen between two plates of dry ice to terminate the reaction, and cyclic AMP content was determined. Both epinephrine and histamine are agonists of the receptors for adenylate cyclase responses. In other experiments, epidermal slices were incubated with 100/ig/ml ofcholera toxin and 1 mM IBMX for 3 h, or with 100 /IM forskolin and 1 mM IBMX for 1 h. in the
30
20
1.4.5'trisphosphate
Slices of epidennal sheets ( 5 x 1 0 mm) were pre-incubated in RPMI 1640 medium for 20 min at 37°C in a water bath, and then incubated in fresh medium with 50 ;^M of substance P. The tissues were rapidly frozen on dry ice after 10-120 s incubation.'" Otherwise the epidermal sheets were incubated with different concentrations of substance P for 30 s. The samples were then homogenized with 4 N perchloric acid and kept on ice for 20 min. Proteins were sedimented by centrifugation at 2000 g for 15 min at 4°C. Supernatants were titrated to pH 7- 5 with 7- 5 mM HEPES. 1 • S4 mM KOH. and kept on ice for 30 min. Precipitated KCIO4 was sedimented and removed. The supernatant was used as samples for IP5.
e: 10
0
0
30
60
90
120
Time ( s ) Figure 1. The time course ofthe effect of substance P on inositol 1,4,Strlsphosphiite accumulation in pig epidermis. Pig epidermal sheets were incubated in KPMI I 640 medium with 50 /JM of substance P at 37°C in a water-bath. The IP? contents were measured as described in the text. Data are expressed as means of triplicate samples. Essentially identical resuits were obtained by three different experiments.
EFFECTS OF SUBSTANCE P IN EPIDERMIS
Table 1. Effects of spantide on inositol 1.4,5-trisphosphate accumulation by substance P
Inositol 1,4,5-trisphosphate (pmol/mg protein) Cuntrol 50 /(M substance P 50 fiM spantide + 50 /IM substance P
025
Data are expressed as means ± 1 SK of quadruple samples. The diflerence between control and spantide + substance P levels is not statistically significant.
presence or absence of SO {.iwt of substance P. The incubation was terminated as described above. Forskolin is a direct activator of adenylate cyclase. Cholera toxin causes accumulation of cyclic AMP through GPT binding protein. Cyclic AMP content in these skin slices was measured by radioimmunoassay using a Yamasa cyclic AMP assay kit. as previously described.'" The protein content was determined using the method of Lowry et «/.'^
597
tion was a rapid and transient process reaching its peak at 20 s. then gradually decreasing (Fig. 1). Pretreatment ofthe epidermis with 50 fm spantide for S min inhibited the effect of suhstance P on IP, accumulation (P
Substance P induces intracellular calcium increase and translocation of protein kinase C in epidermis H.KOIZUMI, H.TANAKA. T.FUKAYA AND A.OHKAWARA Department of Dermatology. Hokkaido University School of Medicine, Kita I 5 Nishi 7, Kita-Ku, Sapporo, 060, japan Accepted for publication JO April 1 992
Summary
Substance P is a neuropeptidc present in, and released from, peripheral C nerve endings. The presence of substance P-positive nerve fibres in the epidermis has been reported. We investigated the efTect of substance P on the transmembrane signalling system of pig epidermal keratinocytes. Treatment of pig epidermis with substance P resulted in an increase in inositol 1,4,5-lrisphosphate (IPs), and in intracellular free calcium. The treatment also resulted in translocation of protein kinase C from a cytosol to a membrane fraction. Substance P, however, did not affect the /i-adrenergic- or histamine (H2)- adenylate cyclase responses of the epidermis. Neither forskolin-induced. nor cholera toxininduced cyclic AMP accumulation were affected by substance P treatment. These results are consistent with the view that substance P stimulates phosphatidylinositol-4.S-bisphosphate (PlPi) hydrolysis of keratinocytes. resulting in IPj-Ca-* and diacylglycerol-protein kinase C signal activation. Although protein kinase C is known to affect the epidermal adenylate cyciase system, no evidence for such 'cross-talk regulation' was detected in keratinocytes by substance P treatment.
Substance P is a neuropeptide. which is present in unmyelinated nerve endings in the skin.'^ Substance Ppositive nerve endings have been demonstrated in the epidermis and dermis of normal and psoriatic skin, and psoriatic plaques are more densely innervated than normal skin.' The content of substance P is also reported to be elevated in psoriatic skin.'* Substance P is released from peripheral nerve endings by various stimuli. Farber et aV suggested that substance P might have pathophysiological significance in psoriasis. According to their view, substance P released from the peripheral nerve endings afTects various cell systems directly or indirectly, resulting in the development of psoriatic lesions. For example, substance P induces histamine release from mast cells, which results in cutaneous vasodilatation.'' and Tanaka et al7 reported that substance P increases Pam 212 keratinocyte proliferation. Although the latter finding has not been confirmed unequivocally by studies using normal human keratinocytes in culture."^ these findings could be related to the formation of the erythematous. hyperproliferative lesions in psoriasis. There is little additional information about other biological aspects of the effects of substance P on keratinocytes. Interestingly, substance P has been shown to stimulate phosphatidylinositol turnover and to Correspondence: Dr H.Koizumi.
activate protein kinase C in other cell systems.'" " The part played by these signalling systems in epidermal proliferation and differentiation, which are deranged in various pathological conditions ofthe epidermis, including psoriasis,''" is a matter of interest. Consequently, we investigated the effect of substance P on various transmembrane signalling systems in epidermal keratinocytes.
Methods Chemicals
Substance P. 12-o-tetradecanoylphorbol-1 3-acetate (TPA). phosphatidylserine. and cholera toxin were purchased from Sigma Chemical Co. (St Louis. MO. U.S.A.). (y-'^P)ATP and D-myo-inositol 1.4.5-trisphosphate (IPO [*H] assay system were purchased from Amersham |apan Corp. (Tokyo. Japan). Spantide was purchased from Bachem Inc. (CA. U.S.A.). Forskolin was obtained from Calbiochem Co. (La JoUa, CA. U.S.A.). Cyclic AMP assay kit is a product of Yamasa Shoyu Co. (Tokyo, Japan). Protein kinase C preparation
Skin slices were obtained from the backs of domestic pigs (weight 5-10 kg) with a Castroviejo keratome set at 0 2 595
596
H.KOIZUMI et al.
mm thickness. The skin slices were treated with 1000 U/ ml of dispase in RPMI 1640 medium for 30 min at 3 7°C. Following the dispase treatment, the pure epidermal sheets were peeled off with sharp forceps. The epidermal sheets were then floated on fresh RPMI 1640 medium for 30 min at 37°C. and were incubated with 50 fiM of substance P for 15 min. Following incubation, the epidermal sheets were wiped with filter paper, and immediately homogenized in a conical glass homogenizer with 5 vol (w/v) of 20 mM Tris HCl. pH 7-5, containing 0 25 M sucrose. 2 mM EDTA. 0-5 mM ECTA. and 2 mM phenylmethylsulphonyl fluoride (PMSF). The enzyme source was centrifuged at 105.000 g for 60 min. The supernatant and the pellet were used as the source of protein kinase C in the cytosol fraction and in the membrane fraction, respectively. These crude extracts were applied to a UE 52-cellulose column and the enzyme was eluted with 1 5 column volumes of 0-08 M NaCl. Protein kinase C activity was measured as previously described.'^
Detection of intracellular calcium alteration
Alteration of intracellular Ca-^^ of single living epidermal keratinocytes was measured by the method of Williams et al.^'' using a fluorescent Ca^+-sensitive dye. Fura 2AM and inverted fluorescence microscopy, as previously described.^^ Pure epidermal sheets were pre-incubated for 30 min. and then incubated with Fura 2-AM for 30 min. The keratinocytes were placed on the stage of an inverted fluorescence microscope. Fifty micromoles of substance P and 1 mM of histamine were added to the cells in succession. Five points of different cells were recorded simultaneously.
Measurement of inosito!
IPj content in the samples was measured by a radiochemical method using a D-myo-inositol 1.4.5-trisphosphate assay system. Ci/(7ic AMP accumulation
Pig epidermal slices obtained by dispase treatment were cut into pieces 5 mm^ with a razor blade, and were rinsed three times in RPMI 1640 medium. They were then floated on the buffer with the keratin layer uppermost. The epidermal slices were pre-incubated at 37°C for 20 min in RPMI 1640 medium in the water bath. For receptor-stimulated cyclic AMP accumulation. the specimens were incubated at 37°C for 5 min in the RPMI 1640 medium with 50/iM epinephrine or 0-1 mM histamine, in the presence or absence of 50 fiM of substance P. The slices were quickly frozen between two plates of dry ice to terminate the reaction, and cyclic AMP content was determined. Both epinephrine and histamine are agonists of the receptors for adenylate cyclase responses. In other experiments, epidermal slices were incubated with 100/ig/ml ofcholera toxin and 1 mM IBMX for 3 h, or with 100 /IM forskolin and 1 mM IBMX for 1 h. in the
30
20
1.4.5'trisphosphate
Slices of epidennal sheets ( 5 x 1 0 mm) were pre-incubated in RPMI 1640 medium for 20 min at 37°C in a water bath, and then incubated in fresh medium with 50 ;^M of substance P. The tissues were rapidly frozen on dry ice after 10-120 s incubation.'" Otherwise the epidermal sheets were incubated with different concentrations of substance P for 30 s. The samples were then homogenized with 4 N perchloric acid and kept on ice for 20 min. Proteins were sedimented by centrifugation at 2000 g for 15 min at 4°C. Supernatants were titrated to pH 7- 5 with 7- 5 mM HEPES. 1 • S4 mM KOH. and kept on ice for 30 min. Precipitated KCIO4 was sedimented and removed. The supernatant was used as samples for IP5.
e: 10
0
0
30
60
90
120
Time ( s ) Figure 1. The time course ofthe effect of substance P on inositol 1,4,Strlsphosphiite accumulation in pig epidermis. Pig epidermal sheets were incubated in KPMI I 640 medium with 50 /JM of substance P at 37°C in a water-bath. The IP? contents were measured as described in the text. Data are expressed as means of triplicate samples. Essentially identical resuits were obtained by three different experiments.
EFFECTS OF SUBSTANCE P IN EPIDERMIS
Table 1. Effects of spantide on inositol 1.4,5-trisphosphate accumulation by substance P
Inositol 1,4,5-trisphosphate (pmol/mg protein) Cuntrol 50 /(M substance P 50 fiM spantide + 50 /IM substance P
025
Data are expressed as means ± 1 SK of quadruple samples. The diflerence between control and spantide + substance P levels is not statistically significant.
presence or absence of SO {.iwt of substance P. The incubation was terminated as described above. Forskolin is a direct activator of adenylate cyclase. Cholera toxin causes accumulation of cyclic AMP through GPT binding protein. Cyclic AMP content in these skin slices was measured by radioimmunoassay using a Yamasa cyclic AMP assay kit. as previously described.'" The protein content was determined using the method of Lowry et «/.'^
597
tion was a rapid and transient process reaching its peak at 20 s. then gradually decreasing (Fig. 1). Pretreatment ofthe epidermis with 50 fm spantide for S min inhibited the effect of suhstance P on IP, accumulation (P
