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A novel steroid receptor in human colon
A990
AGA ABSTRACTS
• EVIDENCE FOR NrrRIC OXIDE AS AN NANC INHIBITORY NEUROTRANSMITTER: STUDIES USING MUTANT MICE WITH DISRUPTED ncNOS GENE. H. Mashimo, X. D. He, P. L. Huang, M. C. Fishman, R. K. Goval. Gastrointestinal Unit and Cardiovascular Research Center, Massachusetts General Hosp., Boston, MA and Gastroenterology Dept., Beth Israel Hosp., Boston, MA. Nitric oxide (NO) has been implicated as a mediator of nonadrenergic noncholinergic (NANC) neurotransmission in the gut. However, whether NO is a tru e antegrade neurotransmitter, or serves as a post-junctional mediator of another inhibitory neurotransmitter, is unclear. To elucidate the role of NO, we performed studies using mutant mice produced by targeted disruption of the ncNOS gene by homologous recombination. These mutant mice develop gastric dilation and muscle hypertrophy. Intracellular membrane potentials were recorded from gastric fundic circular muscle strips in the mutant and wild-type mice. Inhibitory junctional potentials (IJP) were evoked by electrical field stimulation of the strips treated with a mixture of guanethidine, atropine, and substance P desensitization. Wild-type mice showed two overlapping tetrodotoxinsensitive components in the LIPs: the fast IJP (2.1_+0.1sec. duration, 9.9+0.8mV amplitude, n=16) inhibitable by apamin, and the slow HP (3.5+0. lsec, 7.4_+0.4mV, n=9) inhibitab!e by either N-nitro L-arginine (LNA) or the VIP antagonist VIP~n. The mutant mice revealed the fast IJP and lacked the slow lYP. The IJP in the mutant mouse was abolished by apamin but was not modified by L-NA or L-arginine treatment. In the wild-type mice, VIP produced hyperpolarization (9.2+0.4mV, n=5), which was abolished after L-NA treatment. In the mutant mice, VIP failed to produce hyperpolarization. NO produced membrane hyperpolarization in both wild-type and mutant mice (ll+0.5mV and 10.5+l.3mV, n=ll). Other studies have shown that ncNOS is co-localized with VIP in the nerve endings but not in the smooth muscles, and the nerve endings possess prejunctional VIP receptors. Taken together, these studies demonstrate: (1) NO is a tree antegrade inhibitory nenrotransmitter, and ncNOS is its source; (2) peptide VIP acts presynapticaUy to induce synthesis and release of NO, and (3) lack of NO inhibitory neuromuscular transmission is associated with gastric dilatation and hypertrophy.
GASTROENTEROLOGY, VoI. IO8, No. 4
Androgen replacement for castrated TGF~ transger~ male mice recovered hepatic turnorigenesi~ T. Matstnnoto. S. Saitoh, 1L Shimoda, IC Takeham, T. Nagamine, M. Mori, T. Takenchi*, G. Merlino** and H.Takagi. The Fast Dept. of Internal Medicine and Molecular Fmdocfinology*, Gunrna University School of Medicine_ Manhashi, JapurL **: ~ t o r y of Molecular Biology, NCI/NIE, Bethesda MI),LLSA Male transgenic mice overexpressing human TGFa(IvlT42) frequently developed hepatocellular adenoma and carcinoma in adult stage and castration suppressed the hepatic tumorigenesis in these mice(Cell. 61:11371146,1990, Cancer Research 52"5172-5117, 1992). In order to clarify the necessity of androgen in hepatic tumorigenesis in TGFct Wansgenic mice, vasectomized male MT42 were supplied with androge n and their tumorigenesis in liver was analyzed. Method: Thirty transgenic mice were divided into 3 groups; no treatment as central, castration and castration with 5 mg-90 day release pellets of 50dehydmx3rtestostoron(51-1T). 5HT-peilets were implanted into caslrated tmnsgenic mice every 3-month interval for up to 15 months Serum testosteron was assayed with mdioimmtmo-assay for these mice, female transgenic mice and nonlransgenic male and female mic~ Hepatocyte growth was ~uL~yzedwith 1~'qA s~.in~ Results 1). Transgenic mice without any treatment developed 1.4 liver tumors per mouse(rF6). Castrated wansgenic mice developed 0.33 liver tumors per mome(n---6) and castration with 5HT replacement did 1.5Jiver tumors per mouse(n=6). Decreased number of mice in every group is due to death of unknown caus~ 2). Serum testosteron level is 29.7ng/mi in transgenic male,l.38ng/ml in non-transgenic male, 0.53ng/ml in castrated wansgenic male and 2.85 ng/ml in SilT replaced transgenic male mice~ In female, 5.47ng/ml ha transgenic mice and 1.19ng/ml in nonUansgenic femal~ 3). ~ index of PQqA in liver and liver tumor is as follows; no treatment; 0.46%8.5% castration; 0.25,%1.7~ and castration with 5HT; 0,6W~53% Conclusion: TGFa enhanced androgen production in transgenic male and female ~ Androgen dependency in hepamcyte gluwth and hepatic tumorigenesity in ~ transgenic mice was proved by the fact that 5HT replacement recovered hepatic tumorigenedty in castrated male TGFa transgenic mic~
A Novel Steroid Receptor In Human Colon.
• IS PACAP AN INHIBITORY NEUROTRANSMITTER IN THE GUINEAPIG TAENIA COLI? ELECTROPHYSIOLOGICAL EVIDENCE. K. MeConalo~ue1, D.J.K. Lyster~, J.B. Furnessl, 1Dept. of Anatomy & Cell Biology, University of Melbom'ne, Parkville 3052, Australia; 2Dept. of Physiology, Monash University, Clayton 3168, Australia.
Brian McNamara, John Cuffe, *GeraldC. O' Sullivan, Brian J. Harvey. Wellcome Trust Cellular Physiology Reasearch Unit, Dept of Physiology., University College Cork and Dept of Surget% Mercy Hospital, Cork and Universi~." College Cork.
The possible role of pituitary adenylyl cyclase activating peptide (PACAP) in gastrointestinal inhibitory neurotransmission has been investigated in the guinea-pig taenia coli, using electrophysio!ogical techniques. PACAP has been previously shown to be' present in enteric neurons, including those that innervate the taenia coli, and to produce relaxation in this tissue. Membrane potential changes of smooth muscle cells were measured in vitro by intracellular recording, in the presence of hyoscine and nifedipine (106M). PACAP-27 caused a dose dependent decrease in membrane potential of the muscle with a maximum hyperpolarisation of about 10 mV at a concentration of 10-6 M. This response was associated with an increase in conductance of approximately two-fold. The hyperpolarisation was abolished by apamin (10"2vl), a calcium-dependent potassium channel blocker, but suramin (10"4M), which blocks receptors for purines in the taenia toll, only reduced the response by approximately 60%. The hyperpolarisation was not reduced by tetrodotoxin (2xl0"rM), suggesting PACAP acts directly on the muscle. The PACAP antagonist PACAP-6-38 (10"rM) had no effect on the hyperpolarisation evoked by exogenously applied PACAP-27. With continued exposure to PACAP, the hyperpolarisation decayed back to resting membrane potential, possibly due to PACAP receptor desensitisation. Inhibitory junction potentials (IJP's) were markedly reduced in amplitude in the period of presumed receptor desensitisation to PACAP-27; this antagonism of transmission was dose dependent. IJP's were abolished by apamin and tetrodotoxin but not affected by suramin, or by PACAP-6-38. This study implies that PACAP is involved in non-adrenergic inhibitory neurotransmission to intestinal muscle, via an apamin sensitive mechanism, and an increased potassium conductance.
Potassium recycling across the basolateral membranes in human colon occurs through a pH-sensitive and ATP-regulated channel (KATP). Aldosterone activates the KATP channel by a protein kinase C-dependant stimulation of Na+/H + exchange at the basolateral membrane. These effects on potassium • recycling are rapid (initiated < lmin ) and do not depend upon RNA transcription or translation. Here we exanlined the steroid specificity of this response. Epithelial sheets were microdissected from surgical specimens of human colon and mounted in Ussing chambers at 37°C. Electrogenic ion transport was quantified by measuring the short circuit current (S.C.C.) required to clamp the transmembrane potential at 0 mV. The apical membrane was bathed in a high K+ solution designed to mimic the intracellular milieu. The basolateral membrane was bathed in a low chloride Krebs solution. When the apical membrane was pemleabilised with the ionnphore nystatin, the short circuit current was generated by pottassium recycling accross the basolateral membrane through a sulphonylurea inhibitable KATP channel. Under these conditions, aldosterone produced an immediate increase in S.C.C. The maximum response of S.C.C. = 13 + 4% (n = 4) above control and was obtained at lnM with half maximal activation at 0.1 nM. The mineralocorticoid fludrocortisone was less effective, with maximal activation of S.C.C. at lp_M (S.C.C. = 4.6 + 0.8% above control, n = 4). The glucocorticoid, cortisol had no effect at either micromola~" (n = 4) or nanomolar concentrations (n = 4). Oestradiol, a naturally occuring sex steroid, produced an immediate activation of K+ recycling (100riM increased S.C.C by 7.3 :i: 0.8% above controk n = 4). The oestradiol effect, like the response to aldosterone and fludrocortisone, was abolished by pretreatmeni of the basolateral membrane with lmM amiloride to inhibit the Na+/H+ exchanger. The type 1 mineralocorticoid receptor antagonist, spironnlactone, had no effect on the early response to aldosterone. The effects of steroids on K+ recycling and Na'~/H + exchange in human colon appears, therefore, to be mediated by a high affinity receptor which is activated at physiological concentrations of ald0sterone and oestradiol. This receptor is phamracologically distinct from the classical mineralocorticoid cytosolic type 1 receptor and may provide a novel pathway for salt retention by aldosterone and oestradi01. Approved by Medical Ethics Committee. Mercy Hospital, Cork and University College Cork. Funded by Astra Phamaaceuticals (Irl) and The WellcofimTrust (UK).
AGA ABSTRACTS
• EVIDENCE FOR NrrRIC OXIDE AS AN NANC INHIBITORY NEUROTRANSMITTER: STUDIES USING MUTANT MICE WITH DISRUPTED ncNOS GENE. H. Mashimo, X. D. He, P. L. Huang, M. C. Fishman, R. K. Goval. Gastrointestinal Unit and Cardiovascular Research Center, Massachusetts General Hosp., Boston, MA and Gastroenterology Dept., Beth Israel Hosp., Boston, MA. Nitric oxide (NO) has been implicated as a mediator of nonadrenergic noncholinergic (NANC) neurotransmission in the gut. However, whether NO is a tru e antegrade neurotransmitter, or serves as a post-junctional mediator of another inhibitory neurotransmitter, is unclear. To elucidate the role of NO, we performed studies using mutant mice produced by targeted disruption of the ncNOS gene by homologous recombination. These mutant mice develop gastric dilation and muscle hypertrophy. Intracellular membrane potentials were recorded from gastric fundic circular muscle strips in the mutant and wild-type mice. Inhibitory junctional potentials (IJP) were evoked by electrical field stimulation of the strips treated with a mixture of guanethidine, atropine, and substance P desensitization. Wild-type mice showed two overlapping tetrodotoxinsensitive components in the LIPs: the fast IJP (2.1_+0.1sec. duration, 9.9+0.8mV amplitude, n=16) inhibitable by apamin, and the slow HP (3.5+0. lsec, 7.4_+0.4mV, n=9) inhibitab!e by either N-nitro L-arginine (LNA) or the VIP antagonist VIP~n. The mutant mice revealed the fast IJP and lacked the slow lYP. The IJP in the mutant mouse was abolished by apamin but was not modified by L-NA or L-arginine treatment. In the wild-type mice, VIP produced hyperpolarization (9.2+0.4mV, n=5), which was abolished after L-NA treatment. In the mutant mice, VIP failed to produce hyperpolarization. NO produced membrane hyperpolarization in both wild-type and mutant mice (ll+0.5mV and 10.5+l.3mV, n=ll). Other studies have shown that ncNOS is co-localized with VIP in the nerve endings but not in the smooth muscles, and the nerve endings possess prejunctional VIP receptors. Taken together, these studies demonstrate: (1) NO is a tree antegrade inhibitory nenrotransmitter, and ncNOS is its source; (2) peptide VIP acts presynapticaUy to induce synthesis and release of NO, and (3) lack of NO inhibitory neuromuscular transmission is associated with gastric dilatation and hypertrophy.
GASTROENTEROLOGY, VoI. IO8, No. 4
Androgen replacement for castrated TGF~ transger~ male mice recovered hepatic turnorigenesi~ T. Matstnnoto. S. Saitoh, 1L Shimoda, IC Takeham, T. Nagamine, M. Mori, T. Takenchi*, G. Merlino** and H.Takagi. The Fast Dept. of Internal Medicine and Molecular Fmdocfinology*, Gunrna University School of Medicine_ Manhashi, JapurL **: ~ t o r y of Molecular Biology, NCI/NIE, Bethesda MI),LLSA Male transgenic mice overexpressing human TGFa(IvlT42) frequently developed hepatocellular adenoma and carcinoma in adult stage and castration suppressed the hepatic tumorigenesis in these mice(Cell. 61:11371146,1990, Cancer Research 52"5172-5117, 1992). In order to clarify the necessity of androgen in hepatic tumorigenesis in TGFct Wansgenic mice, vasectomized male MT42 were supplied with androge n and their tumorigenesis in liver was analyzed. Method: Thirty transgenic mice were divided into 3 groups; no treatment as central, castration and castration with 5 mg-90 day release pellets of 50dehydmx3rtestostoron(51-1T). 5HT-peilets were implanted into caslrated tmnsgenic mice every 3-month interval for up to 15 months Serum testosteron was assayed with mdioimmtmo-assay for these mice, female transgenic mice and nonlransgenic male and female mic~ Hepatocyte growth was ~uL~yzedwith 1~'qA s~.in~ Results 1). Transgenic mice without any treatment developed 1.4 liver tumors per mouse(rF6). Castrated wansgenic mice developed 0.33 liver tumors per mome(n---6) and castration with 5HT replacement did 1.5Jiver tumors per mouse(n=6). Decreased number of mice in every group is due to death of unknown caus~ 2). Serum testosteron level is 29.7ng/mi in transgenic male,l.38ng/ml in non-transgenic male, 0.53ng/ml in castrated wansgenic male and 2.85 ng/ml in SilT replaced transgenic male mice~ In female, 5.47ng/ml ha transgenic mice and 1.19ng/ml in nonUansgenic femal~ 3). ~ index of PQqA in liver and liver tumor is as follows; no treatment; 0.46%8.5% castration; 0.25,%1.7~ and castration with 5HT; 0,6W~53% Conclusion: TGFa enhanced androgen production in transgenic male and female ~ Androgen dependency in hepamcyte gluwth and hepatic tumorigenesity in ~ transgenic mice was proved by the fact that 5HT replacement recovered hepatic tumorigenedty in castrated male TGFa transgenic mic~
A Novel Steroid Receptor In Human Colon.
• IS PACAP AN INHIBITORY NEUROTRANSMITTER IN THE GUINEAPIG TAENIA COLI? ELECTROPHYSIOLOGICAL EVIDENCE. K. MeConalo~ue1, D.J.K. Lyster~, J.B. Furnessl, 1Dept. of Anatomy & Cell Biology, University of Melbom'ne, Parkville 3052, Australia; 2Dept. of Physiology, Monash University, Clayton 3168, Australia.
Brian McNamara, John Cuffe, *GeraldC. O' Sullivan, Brian J. Harvey. Wellcome Trust Cellular Physiology Reasearch Unit, Dept of Physiology., University College Cork and Dept of Surget% Mercy Hospital, Cork and Universi~." College Cork.
The possible role of pituitary adenylyl cyclase activating peptide (PACAP) in gastrointestinal inhibitory neurotransmission has been investigated in the guinea-pig taenia coli, using electrophysio!ogical techniques. PACAP has been previously shown to be' present in enteric neurons, including those that innervate the taenia coli, and to produce relaxation in this tissue. Membrane potential changes of smooth muscle cells were measured in vitro by intracellular recording, in the presence of hyoscine and nifedipine (106M). PACAP-27 caused a dose dependent decrease in membrane potential of the muscle with a maximum hyperpolarisation of about 10 mV at a concentration of 10-6 M. This response was associated with an increase in conductance of approximately two-fold. The hyperpolarisation was abolished by apamin (10"2vl), a calcium-dependent potassium channel blocker, but suramin (10"4M), which blocks receptors for purines in the taenia toll, only reduced the response by approximately 60%. The hyperpolarisation was not reduced by tetrodotoxin (2xl0"rM), suggesting PACAP acts directly on the muscle. The PACAP antagonist PACAP-6-38 (10"rM) had no effect on the hyperpolarisation evoked by exogenously applied PACAP-27. With continued exposure to PACAP, the hyperpolarisation decayed back to resting membrane potential, possibly due to PACAP receptor desensitisation. Inhibitory junction potentials (IJP's) were markedly reduced in amplitude in the period of presumed receptor desensitisation to PACAP-27; this antagonism of transmission was dose dependent. IJP's were abolished by apamin and tetrodotoxin but not affected by suramin, or by PACAP-6-38. This study implies that PACAP is involved in non-adrenergic inhibitory neurotransmission to intestinal muscle, via an apamin sensitive mechanism, and an increased potassium conductance.
Potassium recycling across the basolateral membranes in human colon occurs through a pH-sensitive and ATP-regulated channel (KATP). Aldosterone activates the KATP channel by a protein kinase C-dependant stimulation of Na+/H + exchange at the basolateral membrane. These effects on potassium • recycling are rapid (initiated < lmin ) and do not depend upon RNA transcription or translation. Here we exanlined the steroid specificity of this response. Epithelial sheets were microdissected from surgical specimens of human colon and mounted in Ussing chambers at 37°C. Electrogenic ion transport was quantified by measuring the short circuit current (S.C.C.) required to clamp the transmembrane potential at 0 mV. The apical membrane was bathed in a high K+ solution designed to mimic the intracellular milieu. The basolateral membrane was bathed in a low chloride Krebs solution. When the apical membrane was pemleabilised with the ionnphore nystatin, the short circuit current was generated by pottassium recycling accross the basolateral membrane through a sulphonylurea inhibitable KATP channel. Under these conditions, aldosterone produced an immediate increase in S.C.C. The maximum response of S.C.C. = 13 + 4% (n = 4) above control and was obtained at lnM with half maximal activation at 0.1 nM. The mineralocorticoid fludrocortisone was less effective, with maximal activation of S.C.C. at lp_M (S.C.C. = 4.6 + 0.8% above control, n = 4). The glucocorticoid, cortisol had no effect at either micromola~" (n = 4) or nanomolar concentrations (n = 4). Oestradiol, a naturally occuring sex steroid, produced an immediate activation of K+ recycling (100riM increased S.C.C by 7.3 :i: 0.8% above controk n = 4). The oestradiol effect, like the response to aldosterone and fludrocortisone, was abolished by pretreatmeni of the basolateral membrane with lmM amiloride to inhibit the Na+/H+ exchanger. The type 1 mineralocorticoid receptor antagonist, spironnlactone, had no effect on the early response to aldosterone. The effects of steroids on K+ recycling and Na'~/H + exchange in human colon appears, therefore, to be mediated by a high affinity receptor which is activated at physiological concentrations of ald0sterone and oestradiol. This receptor is phamracologically distinct from the classical mineralocorticoid cytosolic type 1 receptor and may provide a novel pathway for salt retention by aldosterone and oestradi01. Approved by Medical Ethics Committee. Mercy Hospital, Cork and University College Cork. Funded by Astra Phamaaceuticals (Irl) and The WellcofimTrust (UK).